Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Horvath, Riley Micheal
Doctor of Philosophy - PhD
University of British Columbia
Despite decades of research, HIV-1 remains an ever-prevalent burden on global human health. For many, antiretroviral therapy (ART) is capable of controlling viral replication. However, ART is not a cure and must be administered life-long with discontinuation resulting in viral rebound. The persistence of HIV-1 is the result of a fraction of provirus establishing latency early upon infection of T cells. This population of transcriptionally silent provirus are undetectable to the immune system, unaffected by ART, and are capable of spurious reactivation and clonal proliferation. It has been hypothesized that latency modulating compounds may produce a sterilizing cure for HIV-1. In this thesis, I identify and characterize transcriptional regulators of HIV-1 and examine the ability of small molecules that target these factors to affect viral latency. RBE3 and RBE1 are two of the most highly conserved cis-elements in the HIV-1 LTR. Binding of these elements by the RBF-2 complex, composed of TFII-I and a USF1/USF2 heterodimer, is essential for reactivation of latent provirus. In my work, TFII-I recruits TRIM24 to the LTR resulting in stimulated transcriptional elongation. Furthermore, inhibition of the TRIM24 bromodomain by IACS-9571 reversed proviral latency, an effect that was associated with increased TRIM24 LTR occupancy and facilitation of RNAPII elongation. Thus, TRIM24 bromodomain inhibitors such as IACS-9571 represent a novel class of HIV-1 LRAs that are of interest to the “shock and kill” strategy of viral elimination. Several LTR bound sequence-specific transcriptional activators are regulated by the Mediator complex kinase, CDK8/19. Here, I show that chemical inhibition of CDK8/19 kinase activity enforces the establishment of HIV-1 latency and hinders viral reactivation in response to latency reversal agents. Similar to kinase inhibition, genetic ablation of CDK8 promoted latency while also suppressing reactivation to several agonists. As such, CDK8/19 kinase activity is an attractive latency promoting target that may contribute to the development of a “block and lock” functional cure. Collectively, in this thesis I have identified novel regulators of proviral expression for which existing drugs can target to either promote or antagonize HIV-1 latency thus exhibiting therapeutic potential.
Characterization and exploitation of host factors to modulate HIV-1 latency by Riley Micheal Horvath B.Sc., The University of British Columbia, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2023 © Riley Micheal Horvath, 2023ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Characterization and exploitation of host factors to modulate HIV-1 latency Submitted by Riley Micheal Horvath in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology Examining Committee: Ivan Sadowski, Professor, Biochemistry & Molecular Biology, University of British Columbiaiii Supervisor Thibault Mayor, Professor, Biochemistry & Molecular Biology, University of British Columbia Supervisory Committee Member Eric Jan, Professor, Biochemistry & Molecular Biology, University of British Columbiaiii iiiiiIII Supervisory Committee Member LeAnn Howe, Professor, Biochemistry & Molecular Biology, University of British Columbiaiiiii University Examiner Marc Horwitz, Professor, Biochemistry & Molecular Biology, University of British Columbiaiiii University Examiner David Margolis, MD, Professor, Departments of Medicine, Microbiology & Immunology, Epidemiology, University of North Carolina at Chapel Hill iiiiiiiiiiiiiiiiiiiii External Examiner iii Abstract Despite decades of research, HIV-1 remains an ever-prevalent burden on global human health. For many, antiretroviral therapy (ART) is capable of controlling viral replication. However, ART is not a cure and must be administered life-long with discontinuation resulting in viral rebound. The persistence of HIV-1 is the result of a fraction of provirus establishing latency early upon infection of T cells. This population of transcriptionally silent provirus are undetectable to the immune system, unaffected by ART, and are capable of spurious reactivation and clonal proliferation. It has been hypothesized that latency modulating compounds may produce a sterilizing cure for HIV-1. In this thesis, I identify and characterize transcriptional regulators of HIV-1 and examine the ability of small molecules that target these factors to affect viral latency. RBE3 and RBE1 are two of the most highly conserved cis-elements in the HIV-1 LTR. Binding of these elements by the RBF-2 complex, composed of TFII-I and a USF1/USF2 heterodimer, is essential for reactivation of latent provirus. In my work, TFII-I recruits TRIM24 to the LTR resulting in stimulated transcriptional elongation. Furthermore, inhibition of the TRIM24 bromodomain by IACS-9571 reversed proviral latency, an effect that was associated with increased TRIM24 LTR occupancy and facilitation of RNAPII elongation. Thus, TRIM24 bromodomain inhibitors such as IACS-9571 represent a novel class of HIV-1 LRAs that are of interest to the “shock and kill” strategy of viral elimination. Several LTR bound sequence-specific transcriptional activators are regulated by the Mediator complex kinase, CDK8/19. Here, I show that chemical inhibition of CDK8/19 kinase activity enforces the establishment of HIV-1 latency and hinders viral reactivation in response to latency reversal agents. Similar to kinase inhibition, genetic ablation of CDK8 promoted latency while also suppressing reactivation to several agonists. As such, CDK8/19 kinase activity is an attractive latency promoting target that may contribute to the development of a “block and lock” functional cure. Collectively, in this thesis I have identified novel regulators of proviral expression for which existing drugs can target to either promote or antagonize HIV-1 latency thus exhibiting therapeutic potential. iv Lay Summary HIV/AIDS is an immense burden on global health with over thirty-five million infected. The development of antiretroviral therapy has increased the quality of life of those afflicted but is not a cure and must be administered lifelong. HIV persistence occurs due to the fraction of latently infected T cells. As such, current HIV eradication strategies involve the discovery of compounds capable of modulating HIV latency. The “shock and kill” strategy involves reactivating the HIV virus allowing for the combination of antiretroviral therapy and immune system function to eliminate infected cells. The antithetical strategy is termed “block and lock” and seeks to impose comprehensive silencing that prevents viral reemergence. In this thesis I identify TRIM24 has a host factor that can be chemically targeted to reverse HIV latency. Additionally, I discover that chemical inhibitors of CDK8/19 enforce viral latency and may prove useful in the “block and lock” therapeutic strategy. v Preface Chapter 2 is based on a first author paper published paper. Horvath, R. M., Dahabieh, M., Malcolm, T., Sadowski, I. TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation. Commun Biol. 2023;6(1):86. Malcolm T. performed the yeast-two hybrid screen that identified the TFII-I/ TRIM24 interaction. Dahabieh M. performed the majority of experiments described in Fig. 3 and those in Supplementary Fig. 5. ChIP-seq analysis of ENCODE data was performed by Flibotte S. of the UBC Life Sciences Institute Bioinformatics Facility. Horvath R. M. performed all other experiments. Experimental design was conceived by Horvath R. M. and Sadowski I. Horvath R. M. and Sadowski I. wrote the manuscript. Chapter 3 is based on a first author published paper. Horvath, R. M., Brumme, Z. L., Sadowski, I. Inhibition of the TRIM24 bromodomain reactivates latent HIV-1. Sci Rep. 2023;13(1):556. Brumme Z. L. provided PBMCs from participants with HIV-1 on ART. Horvath R. M. designed and performed all experiments. Horvath R. M. and Sadowski I. wrote the manuscript. Brumme Z. L. provided PBMC samples from individuals on ART. Samples from donor participants were collected with informed consent obtained from all subjects and/or their legal guardian, and handled using methods in accordance with guidelines and protocols approved by the University of British Columbia Clinical Research Ethics Board, certificate H16-02474. Chapter 4 is based on a first author published paper. Horvath, R. M. & Sadowski, I. Upstream Stimulatory Factors Regulate HIV-1 Latency and Are Required for Robust T Cell Activation. Viruses 15, 1470 (2023). Horvath R. M. designed and performed all experiments. Horvath R. M. and Sadowski I. wrote the manuscript. Chapter 5 is based on a first author paper that has been accepted for publication. Horvath, R. M., Brumme, Z. L., Sadowski, I. CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells. 2023. Horvath R. M. designed and performed all experiments. Sadowski I. and Horvath R. M. wrote the manuscript. Brumme Z. L. provided PBMC samples from individuals on ART. Samples from donor participants were collected with informed consent obtained from all subjects and/or their legal guardian, and handled using methods in accordance with guidelines and protocols approved by the University of British Columbia Clinical Research Ethics Board, certificate H16-02474. vi Table of Contents Abstract...................................................................................................................….................... iii Lay Summary.................................................................................................................................. iv Preface..............................................................................................................................................v Table of Contents............................................................................................................................vi List of Figures.................................................................................................................................xi List of Abbreviations..................................................................................................................... xv Acknowledgments........................................................................................................................ xxi Dedication.................................................................................................................................... xxii Chapter 1: Introduction................................................................................................................ 1 1.1 Epidemiology of HIV-1/ AIDS...................................................................................... 1 1.2 HIV-1 genome and virion structure............................................................................... 1 1.2.1 HIV-1 genome............................................................................................. 1 1.2.2 HIV-1 virion structure..................................................................................3 1.3 HIV-1 replication cycle..................................................................................................3 1.3.1 Binding and entry.........................................................................................3 1.3.2 Capsid, reverse transcription, and integration..............................................3 1.3.3 Viral propagation......................................................................................... 5 1.4 HIV-1 latency.................................................................................................................6 1.4.1 Basic overview of transcription................................................................... 6 1.4.2 5’ LTR structure...........................................................................................6 1.4.3 T cell signaling............................................................................................. 7 1.4.4 Tat transactivation....................................................................................... 9 1.4.5 Chromatin structure and epigenetics............................................................9 1.4.6 Chromosomally integrated provirus.......................................................... 11 1.5 Host factors of interest................................................................................................. 12 1.5.1 TFII-I..........................................................................................................12 1.5.2 USF1 and USF2......................................................................................... 13 1.5.3 RBF-2......................................................................................................... 14 1.5.4 TRIM24......................................................................................................14 1.5.5 CDK8 and CDK19..................................................................................... 16 1.6 HIV-1 therapeutic approaches..................................................................................... 16 1.6.1 Antiretroviral therapy.................................................................................16 1.6.2 Individuals cured of HIV........................................................................... 18 1.6.3 Reactivating latent HIV............................................................................. 18 vii 1.6.4 Enforcing deep latency ............................................................................. 19 1.7 Research objectives...................................................................................................... 19 Chapter 2: TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation...................................................................................................................................... 22 2.1 Introduction.................................................................................................................. 22 2.2 Results.......................................................................................................................... 22 2.2.1 TFII-I is required for reactivation of HIV-1 provirus................................ 22 2.2.2 TFII-I interacts with TRIM24.................................................................... 24 2.2.3 TRIM24 activates expression from the HIV-1 LTR.................................. 26 2.2.4 TRIM24 is required for induction of HIV-1 provirus expression..............28 2.2.5 Interaction of TRIM24 with the HIV-1 LTR requires TFII-I.................... 31 2.2.6 TRIM24 stimulates HIV-1 transcriptional elongation............................... 32 2.2.7 Loss of TRIM24 does not cause substantial effects on LTR-associated histone modification...................................................................................35 2.2.8 Regulation of HIV-1 by TRIM24 is independent of TRIM28................... 35 2.2.9 TFII-I and TRIM24 co-regulate specific subsets of cellular genes........... 36 2.3 Discussion.................................................................................................................... 39 2.4 Materials and methods................................................................................................. 41 2.4.1 Cell and virus culture................................................................................. 41 2.4.2 shRNA knockdown.................................................................................... 41 2.4.3 TRIM24 knockout...................................................................................... 42 2.4.4 Immunoblotting and immunoprecipitation................................................ 42 2.4.5 Chromatin immunoprecipitation and quantitative PCR............................. 43 2.4.6 BioID assays.............................................................................................. 44 2.4.7 Luciferase reporter assays.......................................................................... 44 2.4.8 Q-RT-PCR................................................................................................. 44 2.4.9 ChIP-seq analysis....................................................................................... 44 2.4.10 RNA-seq.................................................................................................... 45 2.4.11 Cell adherence assay.................................................................................. 45 2.4.12 Flow cytometry.......................................................................................... 45 2.4.13 Statistics and reproducibility......................................................................45 2.4.14 Data availability......................................................................................... 46 2.5 Chapter acknowledgements......................................................................................... 46 2.6 Supplementary figures................................................................................................. 46 Chapter 3: Inhibition of the TRIM24 bromodomain reactivates latent HIV-1..................... 60 3.1 Introduction.................................................................................................................. 60 3.2 Results.......................................................................................................................... 60 3.2.1 The TRIM24 bromodomain inhibitor IACS-9571 promotes HIV-1 expression.................................................................................................. 60 viii 3.2.2 IACS-9571 stimulates HIV-1 expression in primary CD4+ T cells ex vivo............................................................................................................ 63 3.2.3 IACS-9571 produces synergistic effects with various LRAs.................... 64 3.2.4 TRIM24 is necessary for activation of HIV-1 expression, including by IACS-9571................................................................................................. 67 3.2.5 PROTAC-induced degradation of TRIM24 inhibits HIV-1 expression.... 68 3.2.6 The C-terminal TRIM24 bromodomain is dispensable for activation of HIV-1 expression....................................................................................... 70 3.2.7 TRIM24 LTR occupancy is enhanced by IACS-9571............................... 71 3.2.8 IACS-9571 stimulates HIV-1 transcriptional elongation.......................... 72 3.2.9 Effect of IACS-9571 on HIV-1 provirus expression in CD4+ T-cells from individuals on ART.................................................................................... 74 3.2.10 IACS-9571 partially suppresses T cell activation...................................... 76 3.3 Discussion.................................................................................................................... 78 3.4 Materials and methods................................................................................................. 79 3.4.1 Cell and virus culture................................................................................. 79 3.4.2 Immunoblotting..........................................................................................80 3.4.3 Chromatin Immunoprecipitation................................................................80 3.4.4 Luciferase reporter assays.......................................................................... 80 3.4.5 Q-RT-PCR................................................................................................. 80 3.4.6 Flow cytometry.......................................................................................... 81 3.4.7 Quantitative analysis of IACS-9571 and latency reversing agent interaction.................................................................................................. 81 3.4.8 Statistics and reproducibility......................................................................81 3.4.9 Data availability......................................................................................... 82 3.5 Chapter acknowledgements......................................................................................... 82 3.6 Supplementary figures................................................................................................. 82 Chapter 4: Upstream Stimulatory Factors regulate HIV-1 latency and are required for robust T cell activation................................................................................................................ 87 4.1 Introduction.................................................................................................................. 87 4.2 Results.......................................................................................................................... 87 4.2.1 Regulation of HIV-1 expression by USF1 and USF2................................87 4.2.2 Effect of USF factors on HIV-1 reactivation in response to latency reversing agents and Tat............................................................................ 91 4.2.3 USF2 stabilizes USF1 protein.................................................................... 93 4.2.4 HIV-1 LTR binding of USF proteins......................................................... 95 4.2.5 Global regulation of transcription by USF1 and USF2 in unstimulated T cells............................................................................................................ 98 ix 4.2.6 USF1 and USF2 cooperatively regulate global gene expression in activated T cells....................................................................................... 100 4.2.7 USF1 and USF2 facilitate T cell activation............................................. 102 4.2.8 USF2 deficient cells are refractory towards HIV-1 infection.................. 104 4.2.9 USF2 mediates the establishment of latency upon HIV-1 infection....... 106 4.3 Discussion.................................................................................................................. 108 4.4 Materials and methods............................................................................................... 111 4.4.1 Cell and virus culture............................................................................... 111 4.4.2 Immunoblotting........................................................................................111 4.4.3 Luciferase expression assays................................................................... 111 4.4.4 Transfection assays.................................................................................. 111 4.4.5 Flow cytometry........................................................................................ 111 4.4.6 Generation of USF1 and USF2 knockout clonal lines............................. 112 4.4.7 shRNA mediated knockdown.................................................................. 112 4.4.8 ChIP-qPCR.............................................................................................. 112 4.4.9 RT-PCR................................................................................................... 113 4.4.10 RNA-Seq.................................................................................................. 113 4.4.11 Statistics and reproducibility....................................................................113 4.4.12 Data availability....................................................................................... 113 4.5 Chapter acknowledgments......................................................................................... 114 4.6 Supplementary figures............................................................................................... 114 Chapter 5: CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells...................................................................................................................................... 122 5.1 Introduction................................................................................................................ 122 5.2 Results........................................................................................................................ 122 5.2.1 Chemical inhibition of CDK8/19 kinase activity suppresses HIV-1 expression................................................................................................ 122 5.2.2 CDK8/19 antagonizes HIV-1 reactivation by latency reversing agents....................................................................................................... 125 5.2.3 Effect of CDK8/19 inhibition on cell growth.......................................... 127 5.2.4 Inhibition of CDK8/19 encourages establishment of HIV-1 latency.......128 5.2.5 CDK8/19 inhibitors encourage HIV-1 latency in primary CD4+ T cells ex vivo........................................................................................................... 131 5.2.6 Senexin A and BRD6989 do not inhibit HIV-1 Tat, and block HIV reactivation in a cell-specific manner...................................................... 133 5.2.7 Inhibition of CDK8/19 inhibits recruitment of RNA PolII, but promotes association of the mediator kinases with the LTR................................... 135 5.2.8 CDK8 and CDK19 are activators of HIV-1 expression...........................137 5.2.9 CDK8/19 inhibitors suppress HIV-1 reactivation in CD4+ T cells..........141 5.3 Discussion.................................................................................................................. 143 x 5.4 Materials and methods............................................................................................... 144 5.4.1 Cell culture, virus culture, and lentiviral transduction............................. 144 5.4.2 shRNA knockdown.................................................................................. 145 5.4.3 CDK8 knockout....................................................................................... 145 5.4.4 Immunoblotting........................................................................................146 5.4.5 Chromatin immunoprecipitation.............................................................. 146 5.4.6 Luciferase reporter assays........................................................................ 146 5.4.7 RT-PCR................................................................................................... 146 5.4.8 Flow cytometry........................................................................................ 147 5.4.9 Statistics and reproducibility....................................................................147 5.4.10 Data availability....................................................................................... 147 5.5 Chapter Acknowledgements...................................................................................... 147 5.6 Supplementary figures............................................................................................... 148 Chapter 6: Conclusion............................................................................................................... 156 6.1 TRIM24 facilitates HIV-1 transcriptional elongation................................................156 6.2 TRIM24 can be targeted by bromodomain inhibitors to reactivate latent HIV-1......157 6.3 Role of the Upstream Stimulatory factors for HIV-1 expression and the T cell inflammatory response............................................................................................... 157 6.4 Chemical inhibitors of CDK8/19 enforce HIV-1 latency in CD4+ T cells................ 158 6.5 Closing remarks......................................................................................................... 159 References................................................................................................................................... 161 xi List of Figures Figure 1.1 HIV-1 genome organization........................................................................................... 2 Figure 1.2 HIV-1 replication cycle.................................................................................................. 5 Figure 1.3 Organization of the HIV-1 5’ LTR................................................................................ 7 Figure 1.4 T cell signaling pathways............................................................................................... 8 Figure 1.5 Overview of the latent and transcriptional active 5’ LTR............................................ 11 Figure 2.1 TFII-I promotes HIV-1 expression............................................................................... 23 Figure 2.2 TRIM24 interacts with TFII-I...................................................................................... 25 Figure 2.3 HIV-1 LTR transcription is activated by TRIM24....................................................... 27 Figure 2.4 TRIM24 is an activator of HIV-1 expression in T cells............................................... 29 Figure 2.5 TRIM24 knockout encourages establishment of immediate latency............................ 30 Figure 2.6 TFII-I/ TRIM24 co-localize to the HIV-1 LTR........................................................... 32 Figure 2.7 TRIM24 stimulates elongation of HIV-1 transcription................................................ 34 Figure 2.8 Loss of TRIM24 does not induce restrictive LTR chromatin organization................. 35 Figure 2.9 TFII-I enhances the TRIM24 transcriptional program................................................. 38 Figure 2.10 TRIM24 is an RBF-2 cofactor that stimulates transcriptional elongation of HIV-1............................................................................................................................................. 41 Figure S2.1 Effect of T cell activation on TFII-I and TRIM24 expression................................... 46 Figure S2.2 TurboID fusion proteins expressed in HEK293T cells.............................................. 47 Figure S2.3 TRIM24 knockout inhibits HIV-1 expression............................................................ 47 Figure S2.4 NFκB p65 is recruited to the LTR upon T cell activation.......................................... 48 Figure S2.5 TRIM24 and TFII-I co-localize to the LTR in HeLa cells......................................... 48 Figure S2.6 TRIM24 depletion does not alter CDK9 or cyclin T protein levels........................... 49 Figure S2.7 CDK9 kinase activity is necessary for induction of HIV-1 expression..................... 49 Figure S2.8 TRIM24 regulates HIV-1 latency independently of TRIM28................................... 50 Figure S2.9 Co-localization of TRIM24 and TFII-I in K562 cells................................................ 51 Figure S2.10 TRIM24 KO and TFII-I knockdown differentially expressed genes (DEG)........... 52 xii Figure S2.11 Loss of TRIM24 or TFII-I does not prevent global T cell activation...................... 53 Figure S2.12 TRIM24 KO gene regulation is reflected by TFII-I knockdown.............................. 54 Figure S2.13 TRIM24 regulated cellular adhesion genes are similarly affected by TFII-I knockdown..................................................................................................................................... 55 Figure S2.14 Representative flow cytometry gating strategy........................................................ 56 Figure S2.15 Full length western blots.......................................................................................... 57 Figure 3.1 IACS-9571 promotes HIV-1 expression...................................................................... 62 Figure 3.2 IACS-9571 regulates HIV-1 latency in primary CD4+ T cells.....................................64 Figure 3.3 IACS-9571 produces activation in combination with T cell signaling agonists.......... 66 Figure 3.4 IACS-9571 targets TRIM24 to induce expression of HIV-1....................................... 68 Figure 3.5 Effect of dTRIM24 on HIV-1 expression.................................................................... 69 Figure 3.6 TRIM24 chromatin binding is dispensable for activation of HIV-1 transcription....... 71 Figure 3.7 Association of TRIM24 with the HIV-1 LTR is enhanced upon IACS-9571 treatment........................................................................................................................................ 72 Figure 3.8 IACS-9571 stimulates elongation from the HIV-1 LTR.............................................. 74 Figure 3.9 IACS-9571 and PEP005 synergistically activate HIV-1 expression in PBMCs.......... 75 Figure 3.10 IACS-9571 does not cause T cell activation.............................................................. 77 Figure S3.1 IACS-9571 promotes HIV-1 expression.................................................................... 82 Figure S3.2 Quantitative analysis of synergy between IACS-9571 and latency reversal agents............................................................................................................................................. 83 Figure S3.3 Jurkat Tat Clone #11 is responsive to IACS-9571..................................................... 84 Figure S3.4 Analysis of surface CD69 by flow cytometry............................................................ 85 Figure S3.5 Full length western blots............................................................................................ 86 Figure 4.1 USF2 knockout disrupts HIV-1 expression..................................................................89 Figure 4.2 USF1 and USF2 activate HIV-1 LTR transcription..................................................... 90 Figure 4.3 Role of USF factors for the induction of HIV-1 in response to latency reversal agents and Tat........................................................................................................................................... 92 Figure 4.4 USF1 expression is stabilized in the presence of USF2............................................... 94 xiii Figure 4.5 USF1 and USF2 are capable of localizing independently to the LTR......................... 96 Figure 4.6 Factor determinants of RBF-2 formation..................................................................... 97 Figure 4.7 Transcriptomic effects of USF1 or USF2 knockout under basal conditions................ 99 Figure 4.8 Regulation of the activated T cell transcriptome by USF1 and USF2....................... 101 Figure 4.9 USF1 and USF2 facilitate T cell activation................................................................103 Figure 4.10 T cells deficient of USF2 are resistant to HIV-1 infection....................................... 105 Figure 4.11 USF2 knockout promotes the establishment of latency upon HIV-1 infection........107 Figure S4.1 shRNA knockdown of USF2 suppresses HIV-1 expression.................................... 114 Figure S4.2 USF2 knockout limits proviral induction in TZM-bl cells...................................... 115 Figure S4.3 Reincorporation of USF1 or USF2 rescues LTR transcription in USF2 KO cells.............................................................................................................................................. 116 Figure S4.4 Loss of USF1 or USF2 does not affect cellular growth........................................... 117 Figure S4.5 USF2 regulates HIV expression regardless of Tat.................................................. 118 Figure S4.6 Loss of either USF1 or USF2 hinders expression of genes downstream of multiple T cell activation pathways............................................................................................................... 119 Figure S4.7 USF2 mediates HIV-1 infection of T cells...............................................................120 Figure S4.8 Representative flow cytometry gating strategy........................................................ 121 Figure 5.1 CDK8/19 inhibitors impair reactivation of HIV-1 provirus....................................... 124 Figure 5.2 CDK8/19 inhibitors prevent reactivation of HIV-1 in cell models of HIV-1 latency.......................................................................................................................................... 125 Figure 5.3 Inhibition of CDK8/19 impairs reactivation of HIV-1 by latency reversing agents........................................................................................................................................... 126 Figure 5.4 Effect of Senexin A and BRD6989 on cellular growth and viability......................... 128 Figure 5.5 Inhibition of CDK8/19 promotes establishment of latency in newly infected T cells.............................................................................................................................................. 130 Figure 5.6 HIV-1 latency is regulated by CDK8/19 kinase activity in primary CD4+ T cells.... 132 Figure 5.7 CDK8/19 inhibitors do not affect HIV-1 transactivation by Tat................................ 134 Figure 5.8 Inhibition of CDK8/19 impairs recruitment of RNA Polymerase II to the HIV-1 LTR.............................................................................................................................................. 136 xiv Figure 5.9 Effect of CDK8 gene knockout on PMA induced HIV-1 expression........................ 138 Figure 5.10 CDK8 knockout desensitizes HIV-1 to several latency reversal agents...................140 Figure 5.11 Senexin A inhibits HIV-1 provirus reactivation in PBMCs from patients on ART..............................................................................................................................................142 Figure S5.1 Senexin A and BRD6989 do not affect cell viability at effective concentrations.............................................................................................................................. 148 Figure S5.2 Senexin A and BRD6989 inhibit productive infection in SupT1 cells.................... 149 Figure S5.3 Knockdown of CDK8 expression by shRNA inhibits reactivation of HIV-1 provirus........................................................................................................................................ 150 Figure S5.4 Effect of T cell activation on CDK8/19 expression................................................. 151 Figure S5.5 CDK8 KO and CDK8/19 kinase inhibition differentially impacts HIV-1 expression.................................................................................................................................... 152 Figure S5.6 Example of gating strategy used for flow cytometry............................................... 153 Figure S5.7 Full length western blots.......................................................................................... 154 Figure S6.1 Concluding graphical abstract.................................................................................. 160 xv List of abbreviations ℃ Degree Celsius ✕ g Times gravity AIDS Acquired immunodeficiency syndrome ART Antiretroviral therapy BAF BRG1-associated factor BB B-Box domain bHLH Basic helix loop helix bHLH-LZ Basic helix loop helix leucine zipper BD Bromodomain BET Bromodomain and extra-terminal bp Base pair BRD4 Bromodomain protein 4 CA Capsid CC Coiled-coil domain CCR5 C-C chemokine receptor type 5 CDK7 Cyclin-dependent kinase 7 CDK8 Cyclin-dependent kinase 8 CDK9 Cyclin-dependent kinase 9 cDNA Complementary deoxyribonucleic acid ChIP Chromatin immunoprecipitation CMV Cytomegalovirus Co-IP Co-immunoprecipitation CTD C-terminal domain CTIP2 COUP-TF interacting protein 2 CXCR4 C-X-C chemokine receptor type 4 DAG Diacylglycerol xvi dCA Didehydrocortistatin A DMEM Dulbecco’s modified eagle medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSIF DRB sensitivity-inducing factor DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid eGFP Enhanced green fluorescent protein Env Envelope EZH2 Enhancer of zeste homolog 2 FACS Fluorescence activated cell sorting FBS Fetal bovine serum Gag Group-specific antigen GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFP Green fluorescent protein GO Gene ontology gp glycoprotein GTF General transcription factor HAART Highly active antiretroviral therapy HAT Histone acetyltransferase HDAC Histone deacetylase HDACi HDAC inhibitor HIV Human immunodeficiency virus HMT Histone methyltransferase HMTi Histone methyltransferase inhibitor HP1 Heterochromatin protein 1 HRP Horseradish peroxidase xvii Hrs Hours ICAM Intercellular adhesion molecule 1 IκB Inhibitor-Kappa B IKK Inhibitor kappa B kinase IL2 Interleukin-2 IN Integrase Ion Ionomycin IP Immunoprecipitation IP3 Inositol triphosphate KD Knockdown KO Knockout LAI Prototypical subtype B HIV-1 strain (also known as LAV) LEDGF Lens epithelium-derived growth factor log Logarithm LRA Latency reversal agent LTR Long terminal repeat Luc Luciferase MA Matrix MAPK Mitogen-activated protein kinase mdHIV mini-dual fluorescent HIV-1 LTR reporter MEK MAPK/ER kinase MFI Mean fluorescence intensity mg Milligram MHC Major histocompatibility complex mL Milliliter mM Millimolar mRNA messenger RNA xviii MW (g/mol) Molecular weight (gram/mole) NC Nucleocapsid Nef Negative effector NELF Negative elongation factor NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells NFAT Nuclear factor of activated T-cells ng Nanogram NLS Nuclear localization signal nm Nanometer nM Nanomolar NNRTI Non-nucleoside reverse transcriptase inhibitor NRTI Nucleoside/nucleotide reverse transcriptase inhibitor Nuc Nucleosome PBMC Peripheral blood mononuclear cell PBS Primer binding site PCAF P300/CBP-associated factor PCR Polymerase chain reaction PI3K Phosphatidylinositol 3-kinase PIC pre-integration complex PIP Phosphatidylinositol 4,5- bisphosphate PKC Protein kinase C PLC Phospholipase C PMA Phorbol-12-myristate-13-acetate PR Protease PROTAC Proteolysis targeting chimera P-TEFb Positive transcription elongation factor b qPCR Quantitative PCR xix RasGRP RAS guanyl nucleotide-releasing protein RBE Ras-responsive binding element RBF-2 Ras-responsive element binding factor 2 RBH Red-Blue HIV-1 Rev Regulator of expression of virion proteins RGH Red-Green HIV-1 RING Really interesting new gene domain RNA Ribonucleic acid RNAPII RNA polymerase II RPMI Roswell Park Memorial Institute RRE Rev response element RT Reverse transcriptase RT-qPCR Reverse transcription- quantitative polymerase chain reaction SAHA Suberoylanilide hydroxamic acid SE Standard error Ser2 Serine 2 Ser5 Serine 5 siRNA Small interfering RNA shRNA Short hairpin RNA SIV Simian immunodeficiency virus snRNP Small nuclear ribonucleoprotein Sp1 Specificity protein 1 STAT Signal transducers and activators of transcription SU gp120 Surface glycoprotein 120 SWI/SNF SWItch/Sucrose Non-Fermentable TAR Trans-activation response element Tat Trans-activator of transcription xx TBP TATA-binding protein TCR T cell receptor TE Tris-EDTA TFII-I Transcription factor II-I TM gp41 Transmembrane glycoprotein 41 TNF-𝛼 Tumor necrosis factor-alpha tPIC Transcriptional pre-initiation complex TRIM Tripartite motif tRNA transfer RNA TSA Trichostatin A TSS Transcriptional start site U3 Unique sequence at 3’ end of the genome U5 Unique sequence at 5’ end of the genome USF1 Upstream-stimulatory factor 1 USF2 Upstream-stimulatory factor 2 Vif Viral infectivity factor Vpr Viral protein R Vpu Viral protein U VSV-G Vesicular stomatitis virus-G protein w/v Weight per volume WHO World Health Organization YY1 Yin Yang 1 ZAP-70 Zeta-chain-associated protein kinase 70 µg Microgram µl Microliter µM Micromolar xxi Acknowledgments I would like to thank my research supervisor Ivan Sadowski. As an average academic student, I was unaware of the research profession and am grateful that he took a chance and contacted me about working in the lab. I am thankful to Dr. Sadowski for providing insight, support, and assistance while also giving me the opportunity to work independently. Additionally, I would also like to thank the members of my supervisory committee, Eric Jan and Thibault Mayor for their advice, critiques, and perspectives on my work. I would also like to thank my colleagues at the University of British Columbia with a special thanks to Samantha Peeters, Thomas Dixon-McDougall, and Amanda Ha. Activities such as the beach, Elwoods, and AYCE sushi made Vancouver and graduate school a much more enjoyable experience. Finally, thanks to my family back in Alberta for their constant support and for taking interest in my work. xxii Dedication To my family and friends. 1 Chapter 1: Introduction 1.1 Epidemiology of HIV/ AIDS The etiological agent of acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus (HIV), arose and developed into a global pandemic starting in 1981 (The Lancet, 2021). As the HIV type-1 strain (HIV-1) spread, a second distinct retrovirus was isolated from west Africa in 1986 and was subsequently named HIV-2 (Clavel et al, 1986). Although HIV-1 and HIV-2 are capable of progressing to AIDS, HIV-2 is characterized as being both less transmissible and less pathogenic than HIV-1 (Hughes & Corrah, 1990) (Nyamweya et al, 2013). It is estimated that HIV-1 accounts for 95% of all HIV cases globally with the United States of America categorizing >99.9% of its cases as HIV-1 (Peruski et al, 2020). Given the comparative rarity and reduced pathogenicity of HIV-2, HIV-1 has generally been the focal point of research and is the subject of this thesis. During the course of the HIV pandemic, 84.2 million people have been infected by the virus and 40.1 million individuals have died of AIDS related illnesses (UNAIDS, 2023). For a multitude of reasons, the burden of HIV is not spread equally across the globe or even within the population demographics of a country. Sub-Saharan Africa accounts for roughly 10% of the global population while simultaneously being home to 53% of the world’s people living with HIV (UNAIDS, 2018), and although the indigenous peoples of Canada comprise roughly 5% of the nation’s population, the group experienced nearly one-fourth of country’s new HIV infections in 2019 (Haddad et al, 2021). Furthermore, men who have sex with men account for close to 40% of the reported HIV cases in Canada and thus, this demographic is overrepresented likewise to the indigenous population (Haddad et al, 2021). In addition to population and spatial heterogenicity, trends in both HIV infection and deaths have shifted over time. For instance, there was an estimated 4.9 million new infections and 3.1 million deaths in 2004 (UNAIDS, 2004), as compared to 1.5 million new infections and 0.65 million deaths in 2021 (UNAIDS, 2022). In general, the number of new infections as well as HIV related deaths have decreased over time. However, the progress of HIV/ AIDS management has slowed in recent years due to a multitude of factors including unprecedented events such as the SARS-CoV2 pandemic response (UNAIDS, 2022). Despite advancement in the education, prevention, and diagnosis and treatment of HIV, the retrovirus remains a significant global health concern. 1.2 HIV-1 genome and virion structure 1.2.1 HIV-1 genome Similar to other retroviruses, the size of the HIV-1 genome is ~10-kb (Coffin et al, 1997). In spite of compact genomes and limited coding capacity, viruses must encode essential proteins, hijack host cell processes, and avoid immunological activity in order to replicate. To produce the proteins vital to its replication, the HIV-1 genome possesses 9 open reading frames that 2 transcribe three classes of mRNA: full-length unspliced ~9-kb, incompletely spliced ~4-kb, and completely spliced ~1.8-kb mRNA (Karn & Stoltzfus, 2012). The unspliced ~9-kb HIV-1 mRNA encodes Gag and Pol, the partially spliced ~4-kb mRNA encodes Env, Vif, Vpr, and Vpu, and the completely spliced ~1.8-kb mRNA encodes Tat, Rev, and Nef. Additionally, the genome is flanked by the 5’ and 3’ long terminal repeat (LTR). The 5’ LTR serves as the viral promoter while the 3’ LTR partially encodes Nef and is involved in termination of transcription (Fig. 1.1). Figure 1.1 HIV-1 genome organization. RNAPII transcription begins in the R region of the 5’ LTR. All HIV-1 transcripts possess the same first 69 nucleotides, however, 4 different donor and 8 different acceptor sites are utilized for alternative splicing generating 9 reading frames. Transcribed Gag, Pol, and Env are translated as polyproteins that are proteolyzed into distinct proteins. Vif, Vpr, and Vpu are produced by incomplete splicing while complete splicing generates Tat, Rev, and Nef species. Collectively, the three classes of HIV-1 mRNA facilitate the production of 15 distinct viral proteins (Coffin et al, 1997) (Frankel & Young, 1998) (Fig. 1.1). Gag (group-specific antigen), Pol (polymerase), and Env (envelope) are translated as polyproteins that are subsequently proteolyzed into individual proteins. Gag is cleaved into four proteins, MA (matrix or p17), CA (capsid or p24), NC (nucleocapsid or p7), and p6; Pol produces three proteins, PR (protease), RT (reverse transcriptase), and IN (integrase); and Env generate two proteins, SU (surface or gp120) and TM (transmembrane or gp41). The proteins that are derived from Gag and Env are structural components that generate the core and outer membrane envelope of the virion while the derivatives of Pol provide vital enzymatic functions and are encapsulated within the virion (Frankel & Young, 1998). HIV-1 encodes 6 additional proteins referred to as accessory or auxiliary factors, Vpu (viral protein u), Vif (viral infectivity), Vpr (viral protein r), Nef (negative effector), Rev (regulator of virion gene expression), and Tat (trans-activator of transcription). Briefly, Vpu promotes virion release, Vif stimulates reverse transcription while counteracting host anti-virus factors, Vpr mediates nuclear import of the pre-integration complex and regulates G2/M transition, Nef modulates cellular receptors, enhances viral infectivity, interferes with host cell signalling, and regulates cholesterol trafficking, Rev exports unspliced HIV-1 mRNA from the nucleus to the cytoplasm through interactions with the Rev response element (RRE), and Tat 3 is the viral transa-ctivator of transcription that stimulates elongation of LTR bound RNA polymerase II while also playing a role in inhibiting host cell RNA interference (Li et al, 2005). 1.2.2 HIV-1 virion structure The HIV-1 virion is a spherical enveloped capsid of approximately 145 nM in diameter (Briggs et al, 2003). The envelope is composed of a phospholipid bilayer in which the transmembrane protein gp41is inserted. Envelope embedded trimers of gp41 are attached through non-covalent interactions to trimeric gp120 which project outwards from the surface of the envelope (Gelderblom et al, 1989). Within the envelope, p17 forms the matrix, which holds the double cone shaped capsid core that is built up of p24 (Gelderblom et al, 1989). The capsid contains two copies of the viral genome in the form single stranded, positive sense RNA, that are bound tightly by p7 and p6 proteins that protect the RNA genome from nucleases (Briggs et al, 2003) (Chen et al, 2009). Although each copy of the viral RNA genome contains the necessary genetic information for replication, they are faithfully packaged as a dimer. Dimerization of the RNA genome allows for frequent recombination that increases genetic diversity while also providing a backup in the case of one copy being damaged (Moore & Hu, 2009) (Rein, 2019). Additionally, PR, RT, IN, Vif, Vpr, and Nef proteins are located within the capsid and are essential for replication (Frankel & Young, 1998) (Gelderblom et al, 1989). 1.3 HIV-1 replication cycle 1.3.1 Binding and entry HIV-1 infection begins upon fusion of the virion with the host cell membrane, belonging predominately to a CD4+ T cell, macrophage, or dendritic cell. Trimers of gp120 protrude from the HIV-1 envelope. When triggered, these spikes undergo a cascade of conformational changes that lead to the release of the viral core into the host cell cytoplasm. An initial interaction between the CD4 cell surface receptor and gp120 induces a set of conformational shifts in the glycoprotein that results in the exposure of a binding site for a second surface receptor, being one of the two CD4-associated chemokine receptors CCR5 or CXCR4 (Kong et al, 1998) (Rizzuto et al, 1998). Interestingly, CCR5 is preferred during the early stages of infection, with progression resulting in usage switching to CXCR4 in approximately half of individuals with HIV-1 (Connor et al, 1997) (Regoes & Bonhoeffer, 2005). This phenomenon is related to tropism of the HIV-1 strain and involves alterations to the third variable region (V3) of gp120 that can be as small as a single point mutation (Hwang et al, 1991) (Shimizu et al, 1999) (Trujillo et al, 1996) (Tan et al, 2013). The interaction of gp120 with the primary CD4 receptor and a secondary chemokine receptor induces shifts in the structure of gp41 which then mediates fusion of the viral and host cell membranes (Buzon et al, 2010) (Chan et al, 1997) (Weissenhorn et al, 1997). 1.3.2 Capsid, reverse transcription, and integration Following the fusion of virion and host cell membranes, the capsid is uncoated, and the HIV-1 RNA genome is reverse transcribed into double stranded DNA and integrated 4 permanently into the host cell chromatin. The function(s) of the capsid and the process of its uncoating have been a point of contention. Isolation of viral cDNA early upon infection revealed the presence of IN and RT but failed to extract CA (Farnet & Haseltine, 1991) (Fassati & Goff, 2001), while it was found that the capsid disintegrated when the envelope lipid bilayer was stripped away by detergents (Forshey et al, 2002) (Shah & Aiken, 2011) (Welker et al, 2000). These observations suggested that the capsid was uncoated in the cytosol immediately upon infection and prior to reverse transcription. On the contrary, certain CA point mutations cause defects in reverse transcription and blocking reverse transcription impairs capsid uncoating, thus suggesting that these processes are functionally and temporally connected (Cosnefroy et al, 2016) (Francis et al, 2016) (Hulme et al, 2011) (Hulme et al, 2015) (Mamede et al, 2017) (Rankovic et al, 2017) (Yang et al, 2013). Furthermore, interactions of CA with host factors mediate post-entry events as well as early viral replication (Lahaye et al, 2013) (Rasaiyaah et al, 2013) (Sumner et al, 2020) (Wilbourne & Zhang, 2021) (Yamashita & Engelman, 2017), with these processes interrupted by particular CA mutants (Fischer et al, 2019) (Saito & Yamashita, 2021). Collectively, the current evidence indicates that the capsid functions beyond membrane fusion, playing vital roles in evading host antiviral factors, engaging microtubule associated proteins for cytoplasmic transport, enabling efficient reverse transcription and there is even evidence that the capsid may be transported into the nucleus where it facilitates transference to specific subnuclear regions (Reviewed in Müller et al, 2022; Zila et al, 2021). Reverse transcription of the viral RNA genome into double stranded DNA begins in the cytosol and as mentioned above, is a process that is enhanced by the capsid (reviewed in Aiken & Rousso, 2021). The HIV-1 RT protein possesses both RNA dependent DNA polymerase and RNase H enzymatic activities that facilitate this conversion. RT polymerase uses host tRNALys3 as a primer which base pairs approximately 180 nucleotides from the 5’ end of the viral RNA at a site located in the U5 region of the 5’ LTR called the primer binding site (PBS) (Barat et al, 1989) (Huang et al, 1994) (Huang et al, 1996) (Isel et al, 1995). From the PBS, reverse transcription proceeds in the 5’ to 3’ direction, generating ~180 nucleotides of minus-strand DNA. The activity of RNase H subsequently degrades the RNA portion of the RNA - DNA duplex freeing the minus DNA strand for transfer to the complementary RNA sequence located in the 3’ LTR. Using this minus DNA strand that is now located at the 3’ LTR as a primer, reverse transcription proceeds towards the 5’ LTR. An additional series of templated DNA elongations, RNA degradations, primer sites, and strand transfers subsequently occur, resulting in the synthesis of the full-length ~10 kb double stranded HIV-1 DNA genome with flanking 5’ and 3’ LTR regions (Charneau et al, 1992) (Hungnes et al, 1992) (Julias et al, 2001) (Julias et al, 2002) (Panganiban & Fiore, 1988) (van Wamel & Berkhout, 1998) (Yu et al, 1998) (Reviewed in Hu & Hughes, 2012). Following reverse transcription, the pre-integration complex (PIC) is formed by the association of the genomic linear double-stranded cDNA, MA, RT, Vpr, and various host factors including HMG1 and LEDGF/p75 (Arhel et al, 2007) (Farnet & Bushmen, 1997) (Piller et al, 5 2003) (Shun et al, 2007). Microtubules then mediate retrograde trafficking in a process that uses dynein to actively move the PIC towards the nuclear pore complex (McDonald et al, 2002). Once in the nucleus, integrase cuts the host genome and mediates the irreversible insertion of the HIV-1 provirus. This process is assisted by the host transcriptional co-activator LEDGF that improves the efficiency of insertion and also targets the provirus predominately to the 5’ region of actively transcribed genes that are located in gene-rich chromosomes (Lewinski et al, 2005) (Llano et al, 2006) (Schröder et al, 2002). It is possible for the viral DNA genome to remain unintegrated and exist for some time as one- or two-LTR circles (Sloan & Wainberg, 2011). However, the replication capacity of the unintegrated HIV-1 genome is severely limited (Geis & Goff, 2019) (Irwan et al, 2022) (Smith et al, 2021) (Wang et al, 2016). 1.3.3 Viral propagation Following integration, the HIV-1 provirus operates essentially as a cellular gene with flanking 5’ LTR and 3’ LTR operating as promoter and polyadenylation/ termination site, respectively. Behaving as any eukaryotic promoter, the 5’ LTR is dependent upon host cell transcriptional machinery. Recruitment of RNA polymerase II and the transcriptional pre-initiation complex is mediated by transcription factors bound to LTR cis-elements. As such, HIV-1 proviral transcription is tightly coupled to the activity status of host transcription factors. Completely spliced viral mRNA transcripts dominate early in infection, giving way to partially spliced and unspliced mRNA species over time. Virions form at the point that unspliced full-length HIV-1 RNA are transcribed and the necessary viral proteins have been translated, budding from the host cell plasma membrane and propagating infection (Fig. 1.2). 6 Figure 1.2 HIV-1 replication cycle. The HIV-1 virion binds to the CD4/CD3 surface receptor and either CCR5 or CXCR4 co-receptor of a T cell. Fusion of the virion with the plasma membrane releases the capsid, which is uncoated, freeing two + sense HIV-1 RNA genomes. The RNA genome is then reverse transcribed into double stranded DNA, localized to the nuclease, and integrated into the host cell chromatin. Here, the provirus behaves essentially as a cellular gene, co-opting the host transcriptional machinery to drive transcription of its various mRNA as well as full-length RNA genome. In the cytoplasm, the products of proviral transcription form virions which bud from the host cell membrane to propagate infection. 1.4 HIV-1 latency 1.4.1 Basic overview of transcription The integrated HIV-1 provirus behaves as any cellular gene in that it is dependent upon the host transcriptionally machinery for expression. The 5’ LTR serves as the viral promoter and enhancer that recruits the transcription pre-initiation complex (tPIC), composed of general transcription factors and RNA polymerase II (RNAPII). HIV-1 transcription is TFIID dependent and relies on recruitment of this general transcription factor, consisting of TATA-binding protein (TBP) and 13 – 14 TBP-associated factors (TAFs), to a TATA box located 25 bp upstream of the transcriptional start site. Recruitment of TFIIH is thought to be the rate-limiting step in provirus transcription (Kim et al, 2006), using DNA-dependent ATPase/helicase activity to melt the DNA duplex while the CDK7 subunit phosphorylates Ser-5 of the RNAPII carboxy-terminal domain (CTD) heptapeptide repeats. As 5’ LTR transcription commences, RNAPII is associated with DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) and is subject to promoter proximal pausing. Recruitment of P-TEFb (CDK9/Cyclin T1) hyperphosphorylates DSIF, NELF, and RNAPII CTD Ser-2 resulting in stimulation of processive transcriptional elongation (Bourgeois et al, 2002) (Fujinaga et al, 2004) (Isel & Karn, 1999) (Ivanov et al, 2000) (Kim et al, 2002) (Komarnitsky et al, 2000) (Zhang et al, 2007). Processing and polyadenylation of proviral transcripts is mediated by the 3’ LTR, by which several regulatory elements are employed to ensure that the 3’ LTR, not the 5’ LTR, is utilized (Ashe et al, 1995) (Ashe et al, 2000) (Das et al, 1999) (Gilmartin et al, 1995) (Valente et al, 2009). 1.4.2 5’ LTR structure The ~630 bp HIV-1 LTR serves as the viral promoter and is divided into U3, R, and U5 regions (Fig. 1.3A). Conversely, antisense transcription directed from the 3’ LTR is disproportionally weaker due to transcriptional interference (Cullen et al, 1984) (Klaver & Berkhout, 1994) (Michael et al, 1994). Over fifty DNA binding host factors have been observed to interact with the LTR in vitro and/or in vivo (Pereira et al, 2000). However, given that HIV-1 replication is an error prone process (Bruner et al, 2016), the LTR displays a great degree of sequence diversity of which a TATA box, three tandem SP1 sites, two overlapping NFκB/NFAT sites, an E-box, and Ras-response elements 1 and 3 (RBE1 and RBE3) are the most highly 7 conserved cis-elements (Chen et al, 2005) (Estable et al, 1996). Within the HIV-1 core promoter are the TBP associated TATA box, SP1 sites, and the Ras-response element binding factor 2 (RBF-2) bound RBE1 element (Fig. 1.3B). The NFκB/NFAT elements, USF1/USF2 E-box site, and RBF-2 occupied RBE3 element are found within the enhancer and modulatory regions (Fig. 1.3B). These stringently conserved elements are required for efficient replication of HIV-1, an unsurprising feature given the low fidelity of viral replication. Figure 1.3 Organization of the HIV-1 5’ LTR. A: The HIV-1 5’ LTR serves as the viral promoter and is divided into U3, R, and U5 regions. RNAPII transcription begins at the R region and proceeds through the U5. Assembly of the general transcription machinery and RNAPII occurs in the U3 region as mediated by site specific DNA binding transcription factors and associated co-factors. B: The U3 core promoter and enhancer regions contain the most conserved sequences for binding of the RBF-2 complex (TFII-I, USF1/2), NFκB, NFAT, and SP1. 1.4.3 T cell signaling Multiple host transcription factors that associate with the HIV-1 LTR are regulated by T cell signaling, coupling proviral expression to T cell activation. In general, T cell signalling is the process by which extracellular information is propagated to the nucleus where the message alters gene expression, allowing the cell to be dynamic within the larger context of its environment (Fig. 1.4). T cell receptor engagement initiates activation. Contact of CD3 associated CD4 receptor by antigen presenting cells or cytokines such as IL2 induces T cell receptor (TCR) engagement and formation of the supramolecular activation complex (SMAC) (Grakoui et al, 1999) (Monks et al, 1998). SMAC formation stimulates Lck and Zap70 tyrosine kinases which in turn activate phospholipase Cγ (PLCγ) triggering the hydrolysis of phosphatidylinositol 4,5- bisphosphate (PIP) into the secondary messengers diacylglycerol (DAG) and inositol triphosphate (IP3) (Reviewed in Courtney et al, 2018). IP3 and DAG secondary messengers direct distinct cellular pathways. Cytosolic presence of IP3 mobilizes the release of intracellular calcium (Ca2+) causing the phosphatase calcineurin 8 to be activated. Calcineurin dephosphorylates NFAT, revealing a nuclear localization signal that results in NFAT being accumulated in the nucleus where it influences expression of target genes. Meanwhile, DAG triggers the activation of protein kinase C (PKC) pathway and the mitogen activated protein kinase (MAPK) cascade. Tethering of NFκB to inhibitor of kappaB (IκB) retains the protein in the cytoplasm, preventing its role in gene regulation. Activation of PKC stimulates inhibitor of kappa kinase (IKK) which phosphorylates IκB causing it to be degraded. The newly liberated NFκB now translocates to the nucleus where it can affect the expression of target genes. Additionally, DAG activates the Ras-Raf-MEK-ERK MAPK cascade which influences the activity of multiple transcription factors including AP1 and RBF-2 (Reviewed in Courtney et al, 2018; Sadowski & Mitchell 2005). As noted above, the most highly conserved elements on the HIV-1 LTR include sites for NFAT, NFκB, and RBF-2, all of which assume the role of transcriptional activator in the context of HIV-1. As such, HIV-1 has evolved to hijack the downstream effectors of T cell signaling, thus proviral transcription is interconnected to T cell activation state. Figure 1.4 T cell signaling pathways. Engagement of the CD4/CD3 T cell receptor with its cognate antigen stimulates Lck and Zap70 tyrosine kinases leading to the production of IP3 and DAG from PLCγ hydrolysis of PIP. IP3 and DAG are secondary messengers; IP3 causes an increase in calcium activating the calcineurin pathway while DAG triggers PKC and Ras-MAPK cascades. The phorbol ester PMA stimulates Ras and PKC signaling while ionomycin activates the calcineurin pathway. PKC agonists such as the cytokine TNFα or PEP005 prompts the transcription factor NFκB. 9 1.4.4 Tat transactivation High level HIV-1 transcription is facilitated by the virally encoded trans-activator of transcription, Tat, resulting in the formation of a powerful feedback loop (Sodroski et al, 1985) (Sodroski et al, 1985). The stimulatory effect of Tat requires the trans-activation response (TAR) sequence located within nucleotides +1 and +59 of the LTR R region (Muesing et al, 1987). The TAR region is not a typical transcription element as its effects are dependent upon orientation and position. Rather, as it is transcribed, TAR RNA forms a highly stable, nuclease-resistant, stem-loop structure that is recognized by Tat (Berkhout et al, 1989) (Selby et al, 1989). In the absence of Tat, RNAPII transcription complexes accumulate, and transcripts are aborted shortly following initiation (Kao et al, 1987) (Laspia et al, 1989). Through interaction with TAR, Tat functions to bind and recruit P-TEFb, a heterodimer of Cyclin T1 and CDK9, to the LTR, (Tahirov et al, 2010) (Wei et al, 1998) resulting phosphorylation of the pause factors NELF and DSIF, causing the former to dissociate and the latter to be converted into a stimulatory elongation factor (Bourgeois et al, 2002) (Fujinaga et al, 2004) (Yamada et al, 2006) (Zhang et al, 2007). Additionally, CDK9 extensively phosphorylates Ser-2 of the RNAP CTD, relieving LTR promoter proximal pausing and stimulating processive elongation (Kim et al, 2002) (Komarnitsky et al, 2000). As Tat is an activator of HIV-1 transcription that is expressed by the viral promoter, a positive regulatory circuit is created. Conditions that restrict viral transcription limit Tat availability and when a threshold is reached, HIV-1 transcription is dramatically reduced, and latency forms (Karn, 2011) (Siliciano & Greene, 2011). Latent, transcriptionally silent provirus can be reactivated, as such, HIV-1 is capable of being successfully transcribed in the absence of Tat. This process displays stochastic properties and involves regulation by host cell transcription factors and T cell activation (Ho et al, 2013) (Weinberger et al, 2005). In the case of reactivation, the provirus exhibits bursts of low level basal transcriptional activation until a threshold in Tat production is met, spurring high level viral transcription. Furthermore, although Tat is a strong activator of HIV-1 transcription, it does not seem sufficient to prevent immediate latency as transcriptional silent provirus are established in T cells that ectopically expressing Tat (Hashemi et al, 2016). 1.4.5 Chromatin structure and epigenetics Although HIV-1 integrates into host genome, the provirus exerts a level of independent control over its chromatin structure. The 5’ LTR invariably assembles into an ordered nucleosomal structure with the precise positioning of Nuc-0 and Nuc-1 at an upstream enhancer region and the transcription start site, respectively (Sadowski et al, 2008) (Verdin, 1991). As Nuc-1 overlaps the transcription start site, this nucleosome is an impediment to transcription and is evicted upon induction of proviral expression (Fig. 1.5) (Verdin et al, 1993). The observed arrangement of nucleosomes along the LTR is inconsistent with that of being dictated by intrinsic DNA sequence characteristics (Pazin et al, 1996) (Widlak et al, 1997). Rather, protein 10 factors dictate LTR chromatin structure. The BRG1-associated factors (BAF) ATP-dependent SWI/SNF chromatin remodeling complex is recruited to the LTR by the short isoform of BRD4 (BRD4S) where it positions Nuc-1 over the transcription start site. Removal of BRD4S or BAF from the LTR by factor depletion or BRD4 bromodomain inhibition causes eviction of Nuc-1 and increased expression from chromosomally integrated HIV-1 (Conrad et al, 2017) (Rafati et al, 2011). Furthermore, a number of transcription factors including NFκB and RBF-2 (TFII-I, USF1/2) occupy the nucleosome-depleted region that encompasses the LTR enhancer region regardless of proviral transcription state (Fig. 1.5) (Bernhard et al, 2013) (Chen et al, 2005) (Williams et al, 2006). As such, it is possible that binding of the enhancer region by transcription factors prevents the encroachment of nucleosomes (Struhl & Segal, 2013). Typical features of latent HIV-1 are high levels of histone deacetylases (HDACs) causing the accumulation of deacetylated histones (Ylisastigui et al, 2004), as well as the recruitment of histone methyltransferases (HMTs) that establish methylation patterns consistent with that of repressive heterochromatin (Fig. 1.5) (du Chéné et al, 2007) (Pearson et al, 2008) (Tyagi et al, 2010). Specific cis-acting factors mediate the recruitment of HDACs and HMTs to the LTR. YY1 occupies LTR cis-elements located at the transcription start site and approximately -115, where the factor has been shown to recruit HDAC1 (Coull et al, 2000) (He & Margolis, 2002). The LTR of unstimulated proviruses is associated with NFκB p50 homodimers as well as CBF-1, both of which recruit HDAC1 to repress viral expression (Tyagi & Karn, 2007) (Williams et al, 2006). Binding of SP1 to three tandem sites immediately upstream of the transcription start serves as a platform for CTIP2 that nucleates the formation of a heterochromatin complex composed of HP1α, HDAC1, HDAC2, and the SUV39H1 HMT (Fig. 1.5, Non-permissive) (Marban et al, 2005) (Marban et al, 2007). The histone methyltransferases SUV39H1, G9a (EHMT2), and EZH2 (PRC2) are associated with latent provirus where they are responsible for the deposition of H3K9me3, H3K9me2, and H3K27me3, respectively (du Chéné et al, 2007) (Friedman et al, 2011) (Imai et al, 2010) (Nguyen et al, 2017). Although the recruitment of various HMTs to the LTR are ill-defined, CTIP2 as well as the long noncoding RNA MALAT1 have been identified to mediate HMT recruitment (Marban et al, 2005) (Marban et al, 2007) (Qu et al, 2019). Finally, although there have been observations of increased DNA methylation being associated with viral repression (Blazkova et al, 2009) (Kauder et al, 2009), the latent reservoir of either aviremic patients receiving highly active antiretroviral therapy or elite controllers display very limited DNA methylation (Palacios et al, 2012), suggesting that DNA hypermethylation is not a driver of HIV-1 latency. 11 Figure 1.5 Overview of the latent and transcriptional active 5’ LTR. The proviral 5’ LTR is distinctly organized depending on transcriptional activity. In a latent state, the viral promoter is bound by numerous transcription factors that recruit restrictive epigenetic modifying enzymes. Additionally, two nucleosomes, Nuc-0 and Nuc-1, are precisely positioned within an enhancer region and at the transcription start site. Upon induction, the LTR is bound by transcription factors that recruit the basal transcription machinery and epigenetic modifiers that are associated with transcriptional activation, and displacement of Nuc-1. RBF-2, composed of USF1/2 and TFII-I, is maintained on the LTR regardless of transcriptional activity. 1.4.6 Chromosomally integrated provirus In order to contain the organism’s genetic code within the nucleus, the DNA of eukaryotes is packaged into chromatin. The nucleosome is the fundamental unit of chromatin and consists of 147 DNA base pairs wrapped around a histone octamer (Richmond & Davey, 2003). The histone octamer itself is composed of an H3/H4 tetramer flanked by two H2A/H2B dimers (Richmond & Davey, 2003). Gene regulatory activity produces distinct chromatin environments whereby transcriptionally active genes are often associated with relaxed and open euchromatic states while genes that display low to silenced expression are assembled into restrictive 12 condensed heterochromatin (Takizawa et al, 2008). Differential transcriptional activity is associated with distinct epigenetic patterns. DNA methylation and histone post-translational modifications such as methylation of H3K9 and H3K27 are associated with heterochromatin, while histone acetylation and methylation of H3K36 are hallmarks of actively transcribed genomic regions (Mattei et al, 2022) (Rando, 2007). Upon integration into the genome, HIV-1 is subject to the local host chromatin environment. In particular, heterochromatic site selection often generates latent provirus (Jordan et al, 2001) (Jordan et al, 2003). Furthermore, proviruses that maintain productive expression and those that are induced in response to stimulus, tend to be integrated proximal to activation associated histone modifications such as methylation of H3K4, while latent, unresponsive proviruses correlate with sites enriched for repressive chromatin marks including H3K9me3 (Battivelli et al, 2018) (Chen et al, 2017). In addition to epigenetic influence, expression of cellular genes within the vicinity of the provirus may influence latency through transcriptional interference effects (Han et al, 2008) (Lenasi et al, 2008) (Shan et al, 2011). Integration of HIV-1 tends to occur in the 5’ introns of active genes by a process that utilizes host factors including LEDGF (Lewinski et al, 2005) (Llano et al, 2006) (Schröder et al, 2002). Given the semi-random nature of integration, it may be expected that people living with HIV-1 would display great diversity in proviral selection site. However, this is not the case as proviral sequencing and integration site analysis of infected CD4+ T cells isolated from patients on ART have revealed that single integration sites make up large proportions of HIV-1 infections. This relative lack of diversity is the result of homeostatic and antigen-driven clonal proliferation (Bailey et al, 2006) (Maldarelli et al, 2014) (McManus et al, 2019) (Simonetti et al, 2021). Additionally, integration into particular cancer genes including STAT5B, MKL2, and BACH2 are commonly detected in individuals on ART, suggesting post-integration positive selection (Maldarelli et al, 2014) (Wagner et al, 2013) (Wagner et al, 2014). Elite controllers are individuals infected with non-progressing HIV-1 who do not require ART. Interestingly, proviruses analyzed from elite controllers have been found to be primarily integrated in regions characterized by features of heterochromatin indicating that surrounding epigenetics influence replication competence (Woldemeskel et al, 2020). Furthermore, it has been postulated that the immune system of elite controllers is able to recognize and eliminate actively replicating proviruses that integrate into permissible epigenetic regions (Walker-Sperling, 2017). 1.5 Host factors of interest 1.5.1 TFII-I TFII-I is a multifaceted factor, capable of exerting negative or positive effects on transcription, dependent upon promoter context and cell differentiation state (Roy, 2012). Contrary to its namesake, TFII-I is not a general transcription factor and is not required for sequence specific DNA transcription. TFII-I was initially identified bound to the adenovirus 13 major late (AdML) core promoter, as well various transcriptional initiator (Inr) elements (Roy et al, 1991), but was subsequently observed associated with a number of sequence-specific transcription factors, including SRF, STAT1, STAT3, ATF6 and USF1/2 (Roy, 2012). The structure of TFII-I is organized into six directly reiterated putative (helix-loop-helix) HLH motifs, termed I-repeats (Doi-Katayama et al. 2007) that may facilitate protein - protein interactions or interaction with specific DNA cis-elements in cooperation with additional factors (Vullhorst & Buonanno, 2005). Many of these interactions promote activation of transcription, but TFII-I was also shown to cause transcriptional repression through recruitment of HDAC3 and subsequently the PRC2 component SUZ12 (Wen et al, 2003). 1.5.2 USF1 and USF2 Upstream Stimulatory Factor (USF) was initially identified as a transcriptional regulator of the adenovirus major late promoter (AdML), where it was found bound slightly upstream of the transcriptional start site (Carthew et al, 1985) (Miyamoto et al, 1985) (Sawadogo & Roeder, 1985). Shortly thereafter, it was determined that the AdML bound protein was not a single factor but rather that two USFs existed, USF1 and USF2. The distinct USF1 and USF2 proteins were found to have molecular weights of 43 kDa and 44 kDa respectively (Sawadogo et al, 1988), and are expressed from individual genes located on separate chromosomes (Shieh et al, 1993) (Groenen et al, 1996). The USF1 and USF2 basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factors are ubiquitously expressed, although the ratios vary depending on tissue type (Gregor et al, 1990) (Sirito et al, 1992) (Sirito et al, 1994). The USF1 and USF2 are related factors that possess an overall sequence homology of 44% with the C-terminal b-HLH-LZ displaying ~70% conserved identity (Sirito et al, 1994). Given the high degree of sequence homology (Sirito et al, 1994), similarity in AdML DNA binding (Sawadogo, 1988), and the presence of USF1/USF2 heterodimers dominating within the cell (Viollet et al, 1996), the functional differences of the two proteins have often been neglected. The assumption that USF1 and USF2 share identical operation demands further investigation as knockout mice display unique phenotypes resulting most likely from differential gene expression (Sirito et al, 1998). Likewise with other members of the b-HLH transcription factor family, USFs have been observed to bind E-Box elements possessing the core CANNTG sequence such as within the AdML and RBE1 (CAGCTG) (Baxevanis & Vinson, 1993) (Rada-Iglesias et al, 2008). However, USF binding is not strictly limited to E-Boxes as they have been shown to bind sequences including pyrimidine rich Inr elements and the non-canonical E-Box of RBE3 (ACTGCTGA) (Roy et al, 1997) (Estable et al, 1999) (Chen et al, 2005). Binding of certain DNA sequences by the USF factors has been observed to depend on additional factors such as TFII-I (Chen et al, 2005) (Roy et al, 1991). USF proteins are capable of positively or negatively impacting transcription, an effect that is dependent upon gene context and cellular differentiation state (Andrews et al, 2001) (Ismail et al, 1999) (Qyang et al, 1999). The temporal and spatial nature of USF transcriptional effects have likely led to the conflicting reports of the factors possessing tissue protective or deleterious properties. USF1 reportedly inhibits inflammatory 14 NFκB signaling in lung tissue (Tiruppathi et al, 2014) while USF2 levels positively correlate with expression of proinflammatory cytokines in Th17 cells and refractory rheumatoid arthritis (Hu et al, 2020). Furthermore, USF1 plays an important role in UV response and prevention of tumor development (Galibert et al, 2001) (Corre et al, 2004) (Bouafia et al, 2014) while USF1 and USF2 are activators of HOXA9 suggesting a driver role in leukemia (Zhang et al, 2020). Although the mechanism(s) are ill-defined, USF transcriptional regulation has been reported to involve alterations to chromatin organization. The HS4 insulator element of the chicken β-globin locus requires USF binding to retain H3K4me and H3ac and prevent the encroachment of heterochromatin (West et al, 2004). Additionally, activation of lipogenic genes in response to insulin involves USF1 mediated recruitment of BAF60c and subsequent chromatin remodeling (Wang et al, 2013). Indeed, USFs have been found to be signal regulated transcription factors that are modulated through phosphorylation (Horbach et al, 2015). Phosphorylation of USFs is capable of altering both their DNA binding properties and protein-protein interactions (Galibert et al, 1997) (Nowak et al, 2005) (Spohrer et al, 2017). Physiologically, phosphorylation of USF2 by CDK5 is thought to contribute to carcinogenesis (Chi et al, 2019) while CK2 dependent phosphorylation of USF1 causes altered expression of genes involved in metabolism (Lupp et al, 2014) (Spohrer et al, 2017). 1.5.3 RBF-2 Association of USF1, USF2, and TFII-I forms the Ras-response element binding factor 2 (RBF-2) complex that binds the highly conserved RBE3 and RBE1 HIV-1 LTR cis-elements. TFII-I in particular seems to play a vital role for RBF-2 formation and function as USF1 and USF2 have low affinity for RBE3 and RBE1 in its absence in vitro (Chen et al, 2005) (Dahabieh et al, 2011). Mutations to RBE3 and RBE1 prevent induction of chromosomally integrated provirus, however, these mutations abrogate binding of USF1, USF2, TFII-I, and YY1 (Chen et al, 2005) (Malcolm et al, 2007) (Malcolm et al, 2008). Additionally, a less stringently conserved upstream CACGTG E-Box is located at approximately -166 of the LTR and is a binding site for USF1/USF2 heterodimers (d'Adda di Fagagna et al, 1995) (Sieweke et al, 1998). Similar to RBE3/RBE1, mutating this E-Box results in decreased LTR transcription as assessed by transient reporter and in vitro transcription assays (d'Adda di Fagagna et al, 1995) (Sieweke et al, 1998). These observations suggest that TFII-I, USF1, and USF2 are transcriptional activators of HIV-1 expression. Interestingly, T cell activation induced by the phorbol-ester PMA causes extensive phosphorylation of TFII-I, USF1, and USF2, suggesting a possible regulatory mechanism (Chen et al. 2005). However, the role and mechanism(s) of the RBF-2 components for modulating proviral latency remain poorly defined. 1.5.4 TRIM24 Tripartite-Motif containing protein 24 (TRIM24) possesses amino-terminal RBCC (RING, BBox, coiled-coil) domains, consistent with other members of the TRIM protein family, in addition to a Transcription Intermediary Family (TIF1)-defining carboxy-terminal tandem 15 plant homeodomain - bromodomain (PHD-BRD) motif (Appikonda et al, 2016) (Hatakeyama, 2017). TRIM24 has been associated with both oncogenic and tumor suppressive effects, dependent on cellular context; deletion of TRIM24 promotes hepatocellular carcinoma (HCC) in mice, while TRIM24 expression negatively correlates with cancer progression and patient survival rates for multiple human cancers including for breast and prostate (Tsai et al, 2010) (Herquel et al, 2011) (Groner et al, 2016). Although the mechanisms for development and progression of these cancers are ill-defined, it is thought that TRIM24 dependent transcription is involved. Specifically, TRIM24 was characterized as a cofactor for transcriptional activation by various nuclear receptors including for estrogen (ER) and androgen (AR), and it has been proposed that recruitment of TRIM24 to genes regulated by these factors promotes cellular proliferation and tumor growth (Tsai et al, 2010) (Tisserand et al, 2011) (Pathiraja et al, 2015) (Groner et al, 2016). In addition to the role of TRIM24 as a co-activator of DNA sequence specific transcription factors, this protein interacts with H3K4me0/H3K23ac modified histones through its tandem PHD-BRD domains (Tsai et al, 2010) (Groner et al, 2016) (Lv et al, 2017). As such, TRIM24 is capable of reading epigenetic information in to produce transcriptional changes. In addition to TRIM24 being recruited to genomic loci by modified histones, interaction of the factor with chromatin promotes its SUMOylation, which causes global alterations in transcription (Appikonda et al, 2018). However, the overall molecular mechanism(s) by which TRIM24 functions as a co-activator of transcription are yet to be established. As bromodomains are considered to be “druggable” motifs, specific inhibitors that target the TRIM24 bromodomain have been developed (Zhan et al, 2015). Using protein structure guided design, a TRIM24 bromodomain binding compound, IACS-9571, was developed that inhibits the interaction with histone H3K23ac peptides with nanomolar affinity (Palmer et al, 2016). Interestingly, IACS-9571 was found to inhibit cell growth and metastatic invasive potential of glioblastomas from patients (Han & Sun, 2021). These observations support the contention that TRIM24 may contribute to cancer progression through interaction of chromatin through its bromodomain. In addition to being a cofactor of transcription, TRIM24 contains a C-terminal RING domain that possesses E3 ubiquitin ligase activity. TRIM24 has been shown to target p53 for proteasomal mediated degradation (Allton et al, 2009) (Jain et al, 2014). The mechanism by which TRIM24 targets p53 is an auto-regulatory feedback loop. In response to DNA damage, ATM phosphorylates TRIM24 resulting in a decreased affinity for p53, thus stabilizing p53. Accumulation of p53 induces TRIM24 gene expression causing greater ubiquitylation and degradation of p53, terminating the p53 DNA damage response (Jain et al, 2014). Consequently, TRIM24 may regulate cancer progression through effects on genome stability in addition to alterations in global transcriptional regulation (McAvera & Crawford, 2020). Furthermore, TRIM24 is capably of modifying the functions of CBP and TRAF3 by catalyzing K63-linked ubiquitination. TRIM24 mediated K63 ubiquitination of CBP inhibits macrophage polarization 16 (Yu et al, 2019), while this modification of TRAF3 is part of the anti-viral response to VSV infection (Zhu et al, 2020). Given the numerous observations of TRIM24 mediated ubiquitylation, it is perhaps surprising that this E3 ligase activity is yet to have been observed directly regulating transcription. 1.5.5 CDK8 and CDK19 The RNA Polymerase II mediator kinase complex is comprised of CDK8, or its paralog CDK19, cyclin C, and the regulatory factors Med12 and Med13. This complex transiently associates with the mediator, recruited to the core promoter by transcriptional activators (Nemet et al, 2014), and acts to modulate transcription with signal transduction by phosphorylation of sequence specific activators as well as GTFs (Chong et al, 2020). Defined substrates for CDK8/19 include transcriptional activators and GTF proteins, including the RNA Pol II C-terminal domain (CTD) and mediator subunits (Aristizabal et al, 2013) (Gold et al, 1996) (Nemet et al, 2014). Phosphorylation of transcriptional activators by CDK8/19 can have positive or negative effects on transcription, depending on the functional effect of the modification activators (Nemet et al, 2014). Phosphorylation of -catenin TCF/LEF (Firestein et al, 2008), NFB p65 (Chen et al, 2017), and STAT1/3 (Bancerek et al, 2013) by CDK8 enhances their effect on transcriptional activation. In contrast, factors such as notch intracellular domain (NICD), are negatively regulated by CDK8 phosphorylation, which promotes its degradation (Fryer et al, 2004). Consistent with CDK8-dependent phosphorylation of transcriptional activators being capable of influencing positive or negative effects on activity, alterations in genes encoding CDK8, and cyclin C, have been implicated as both oncogenes and tumor suppressors. CDK8 was found to be overexpressed in a variety of cancers, including breast and colorectal carcinomas, and malignant melanomas, where expression is associated with tumor progression (Firestein et al, 2008). In contrast, CDK8 overexpression also produces tumor suppressive effects of cancers promoted by Notch or EGFR signaling (Gu et al, 2013). Furthermore, mutations in MED12, encoding a regulatory subunit of the mediator kinase are frequently associated with various cancers, in particular those dependent upon hormones (Zhang et al, 2020). The relationship between mutations and overexpression of mediator kinase components and cancer progression has spurned development of specific inhibitors of CDK8/19 activity (Zhang et al, 2021) (Yu et al, 2021) (Aboelmagd et al, 2021), and clinical trials involving CDK8/19 inhibitors are underway, including for ER-positive breast cancers and acute myeloid leukemia (Wu et al, 2020). As noted above, CDK8 modulates the activity of -catenin, NFB, STAT1, and STAT3, all of these transcription factors are known to regulate HIV-1 latency. 1.6 HIV-1 therapeutic approaches 1.6.1 Antiretroviral therapy HIV-1 emerged as an immense challenge to global health in 1981 (The Lancet, 2021). Contraction of the retrovirus was associated with an incredibly high fatality rate with less than 17 1% of infected individuals being able to control disease progression and prevent immune system collapse in the absence of the medical interventions that did not exist (Deeks & Walker, 2007) (Hubert et al, 2000). It took until 1987 for the first potential antiretroviral therapy for HIV-1, zidovudine (azidothymidine, AZT), to be approved and deployed. Mechanistically, AZT is a nucleoside reverse transcriptase inhibitor (NRTI), that was shown in a clinical trial to have a positive impact on clinical progression and death (Fischl et al, 1987). In real-world use, it quickly became apparent that AZT monotherapy was not a radical therapeutic capable of restricting the AIDS pandemic. Challenges of early AZT treatment included that it required stringent adherence to protocol, had high toxicity, and demonstrated leaky suppression of viral replication that resulted in the emergence of multiple resistance mutations (Tseng et al, 2015). Furthermore, although AZT could prolong survival, it failed to provide sustained viral suppression and rarely reversed immune function (Pau & George, 2014). Although AZT monotherapy was not a great success story, it did inspire the combinatorial regimens that are used today. In the mid-1990’s, HIV protease inhibitors (PIs) were combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) with great success as this combination was capable of virological suppression of <400 copies ml-1 resulting in dramatic reductions in morbidity and mortality (Palella et al, 1998). The current therapeutic approach to HIV is to “hit hard, hit early” (Ho, 1995) with a cocktail of two or three mechanistically diverse viral replication inhibitors in what is known as the combinatorial antiretroviral therapy (cART) approach. As of 2014, there are twenty-eight available antiretroviral drugs that belong to six different mechanistic classes including PIs (Saquinavir, Indinavir, Ritonavir, etc.) NRTIs (Abacavir, Tenofovir, Emtricitabine, etc.), NNRTIs (Delavirdine, Efavirenz, Etravirine, etc.), CCR5 antagonist (Maraviroc), fusion inhibitor (Enfuvirtide), and integrase strand transfer inhibitors (INSTIs) (Raltegravir, Elvitegarvir, Dolutegravir) (Pau & George, 2014). Today, various health authorities provide therapeutic recommendations for the treatment of HIV-1. For instance, The British Columbia Centre for Excellence in HIV/AIDS provides a therapeutic guideline for initial ART regimens based on a cost-benefit analysis. Here, this group indicates that the preferential treatment is a three pill per day regimen of Atazanavir (PI), Ritonavir (PI), and Abacavir/Lamivudine (NRTI) (The Committee for Drug Evaluation and Therapy, 2023). Likewise, the United States Department of Health and Human Services indicates that the initial ART treatment generally consists of the use of two NRTIs in combination with a third antiviral belonging to one of the following three drug classes: INSTI, NNRTI, or PI (HHS, 2022). Current cART is capable of reducing viremia to undetectable levels, but it does not represent a cure and must be taken lifelong with discontinuation resulting in immediate viral rebound (Chun et al, 2000). This is a consequence of the extremely long-lived viral reservoir of latently infected CD4+ T cells that is established early upon infection, is unaffected by ART, and escapes immune system clearance (Finzi et al, 1997) (Wong et al, 1997) (Joos et al, 2008). 18 Approximately 1 replication competent provirus is harbored per 106 CD4+ T cells (Shan & Siliciano, 2013) and given that the half life of these cells is about 4 years, it has been estimated that ~88 years of stringent ART would be required to cure an individual of HIV-1 (Hill, 2018). 1.6.2 Individuals cured of HIV To date, a total of five people on earth have possibly been cured of HIV. The strategy that has been likely replicated occurred during the treatment of the patient’s leukemia or lymphoma. These cancer treatments proceeded with immune system depletion followed by allogenic transplantation of hematopoietic stem cells from compatible donors expressing the naturally occurring CCR5Δ32Δ32 mutation. CCR5 is the co-receptor for HIV-1 entry and the homozygous CCR5Δ32 mutation prevents CD4+ T cell infection (Allers et al, 2011) (Allers & Schneider, 2015) (Gupta et al, 2019) (Hütter, 2016). It is currently impractical for the processes that cured these individuals to become mainstream treatments. Although stem cell transplants are rather routine, they are still a medical procedure that runs the risk of serious complications. Furthermore, matching people living with HIV to compatible CCR5Δ32Δ32 donors is unlikely to be feasible. Although there are barriers to applying stem cell transplants therapeutically, these cases are encouraging proof of concepts that it is possible to cure HIV if enough infected cells are eliminated and those cells that remain are resistant to infection. 1.6.3 Reactivating latent HIV As stated above, the primary barrier to a cure for HIV-1 is the long-lived population of latently infected CD4+ T cells (Cohn et al, 2020) (Laskey & Siliciano, 2014). Under normal circumstances, foreign pathogens are presented on the cell surface allowing for immune clearance of viral infections (Lucic & Lusic, 2016). As such, latent HIV is invisible to the immune system as no viral macromolecules are being produced. Consequently, the “shock and kill” strategy has been theorized whereby latency reversing agents (LRAs) are employed to induce proviral expression, thereby exposing infected cells to immune system clearance and viral induced apoptosis (Kim et al, 2018) (Margolis & Archin, 2017) (Thorlund et al, 2017). Importantly, the LRA treated individual would remain on ART to prevent viral propagation. HIV-1 expression is coupled to T cell signalling through binding of downstream transcription factors to the 5’ LTR. As such, a number of T cell signalling agonists including but not limited to phorbol 12-myristate 13-acetate (PMA), ingenol-3-angelate (PEP005), and the PI3K/Akt agonist romidepsin are capable of inducing proviral expression in vitro and ex vivo (Sadowski & Hashemi, 2019). However, global T cell activation can manifest a “cytokine storm” otherwise known as cytokine release syndrome, which is a potentially fatal adverse effect (Shimabukuro-Vornhagen et al, 2018). As such, when considering T cell signalling agonists as potential therapeutics, one must balance the capability to induce HIV expression with the magnitude of effect on immune system activation. 19 In addition to T cell signalling, proviral latency is established by a combination of epigenetic mechanisms that are enzymatically catalyzed (Khan et al, 2018). Concordantly, inhibitors of these epigenetic enzymes, histone deacetylase inhibitors (HDACi) and histone methyltransferase inhibitors (HMTi), have been demonstrated to reactivate latent HIV in vitro and ex vivo (Bernhard et al, 2011) (Contreras et al, 2009) (Friedman et al, 2011) (Imai et al, 2010) (Lu et al, 2014) (Reuse et al, 2009). To date, a number of T cell signalling agonists and HDACi’s have been tested in clinical trials. Many of these drugs were shown to increase HIV RNA transcription, however, none succeeded in reducing the size of the infected reservoir (Archin et al, 2012) (Elliott et al, 2014) (Elliott et al, 2015) (Gutiérrez et al, 2016) (Rasmussen et al, 2014) (Søgaard et al, 2015). Additionally, several BET (bromodomain and extra terminal domain) inhibitors (BETi) such as JQ1 have been shown to induce HIV transcription through inhibiting BRD4 and stimulating the recruitment of P-TEFb to the 5’ LTR (Huang et al, 2017) (Li et al, 2013) (Lu et al, 2016) (Zhu et al, 2012). However, no BETi has yet been approved for clinical trial use. 1.6.4 Enforcing deep latency Given the struggles that have been presented by the “shock and kill” strategy, a diametric therapeutic approach of “block and lock” has been proposed. A functional cure would be the permanent suppression of proviral transcription and thus the elimination of HIV propagation in the absence of any treatment. This is the aim of the “block and lock approach” whereby a latency promoting agent (LPA) enforces a state of deep and irreversible latency in the absence of ART, essentially transforming HIV into an endogenous retrovirus. Didehydro-Cortistatin A (dCA) was the first compound identified and examined for potential LPA activity. Originally isolated from the murine sponge Corticium simplex (Aoki et al, 2006), the steroidal alkaloid cortistatin A (CA) was found to inhibit CDK8/19 kinase activity (Cee et al, 2009) (Pelish et al, 2015). Because of its limited availability from nature, analog derivatives of CA were developed by chemical synthesis, including didehydro-Cortistatin A (dCA) (Shi et al, 2009) (Shi et al, 2011). Subsequently, dCA was found to suppress HIV-1 expression by a mechanism proposed to involve inhibition of Tat, the HIV-1 transactivator of transcription, independently from effects involving inhibition of CDK8/19 activity (Kessing et al, 2017) (Mediouni et al, 2019) (Mousseau et al, 2012) (Mousseau et al, 2015). Other more recently identified LPA targets include spironolactone mediated degradation of the TFIIH XPB subunit (Mori et al, 2021) and inhibition of the mTOR complex (Besnard et al, 2016). As of now, no clinical trials assessing the use of LPAs have been performed. 1.7 Research objectives The primary barrier to a cure for HIV-1 is the extremely long-lived population of latently infected CD4+ T cells. In order to produce either a sterilizing or functional cure, the “shock and kill” and “block and lock” therapeutic strategies of latency modulation have been proposed. Although a number of compounds have shown the ability to regulate HIV-1 latency, none have 20 succeeded in reducing the size of the infected CD4+ T cell population. As such, a greater understanding of the mechanisms that generate the latency phenomenon are required. The purpose of this thesis was to identify regulatory mechanisms of HIV-1 latency and apply small molecule compounds that exploit these processes to modulate proviral expression. Using a yeast-two hybrid screen, our lab identified that TFII-I (RBF-2) interacts with the transcription co-factor TRIM24. As mutations to the RBE3/1 LTR cis-elements that abrogate TFII-I binding render integrated provirus unresponsive to T cell activation, I formed the hypothesis that TFII-I functions to promote HIV-1 expression through association with TRIM24. Furthermore, TRIM24 possesses a bromodomain that can be inhibited with the small molecule IACS-9571, suggesting that this factor may be targetable to modulate latency. In this thesis, I use shRNA knockdown and CRISPR-Cas9 techniques to determine that TFII-I and TRIM24 activate HIV-1 transcription. Using various biochemical approaches, I determine that TFII-I recruits TRIM24 to the 5’ LTR, facilitating release of paused RNAPII. Surprisingly, I found that chemical inhibition of the TRIM24 bromodomain potentiates proviral latency reversal. By characterizing the effect of IACS-9571, I determined that bromodomain inhibition causes enrichment of TRIM24 at the HIV-1 LTR and the subsequent stimulation of RNAPII elongation. Here, I identified TRIM24 bromodomain inhibitors as a novel class of latency reversal agents that may be used in the “shock and kill” HIV-1 elimination strategy. As RBF-2 is a complex consisting minimally of TFII-I and a USF1/2 heterodimer and mutations that prevent it from binding the LTR suppress proviral expression, it is reasonable to predict that USF1 and USF2 are activators of HIV-1 transcription. To this end, I found that depletion of USF2 but not USF1 limited HIV-1 expression. Interestingly, loss of USF2 protein resulted in a corresponding decrease of USF1 protein, an effect that was not associated with diminished USF1 mRNA abundance. Moreover, re-introduction of USF1 or USF2 in USF2 null cells was able to rescue HIV-1 expression suggesting that either factor is capable of regulating HIV-1. Further exploration of genome wide transcription effects of the USFs identified a role for T cell activation, a potential explanation as to why cells lacking USF2 are refractory towards HIV-1 infection. Results presented in this section of my thesis expand our understanding of the Upstream Stimulatory Factors and confirm their role as activators of HIV-1 transcription. Lastly, the transcription factors -catenin TCF/LEF, NFB p65, and STAT1/3 are involved in HIV-1 expression while they themselves are regulated by CDK8/19 dependent phosphorylation. As such, it is possible that the Mediator complex kinase modulates proviral latency. In this section of my thesis, I observed that small molecule inhibitors of CDK8/19 antagonize reactivation of latent HIV-1 in both cell line models and in CD4+ PBMCs isolated from individuals on ART ex vivo. Genetic ablation of CDK8 similarly resulted in decreased proviral expression. Intriguingly however, loss of CDK8 did not mirror kinase inhibition in response to several latency reversal agents suggesting that there are key distinctions between CDK8 and CDK19 for regulation of HIV-1. In this section, I identify CDK8/19 as attractive targets for the development of a “block and lock” functional cure for HIV-1. Collectively, my 21 work has identified two novel drug classes, one with implications for “shock and kill” and the other for “block and lock”, while also expanding our mechanistic understanding of several transcription factors and co-factors. 22 Chapter 2: TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation 2.1 Introduction Advances in antiretroviral therapy (ART) has eliminated HIV/AIDS as a leading cause of death globally, but this treatment does not represent a cure because of the establishment of a long-lived reservoir of latently infected cells which develops in afflicted individuals and is unaffected by ART (Finzi et al, 1997) (Wong et al, 1997) (Joos et al, 2008). Expression of HIV-1 from the 5’ long terminal repeat (LTR) is tightly linked to T cell activation, through the function of factors that bind the LTR enhancer region, regulated by T cell signaling (Brooks et al, 2003). The Ras-response elements (RBE3 and RBE1), located flanking the LTR enhancer region, were identified as required for response to activated Ras signaling (Bell & Sadowski, 1996), and represent the most highly conserved LTR cis elements in provirus from patients that develop AIDS (Estable et al, 1996). These conserved elements bind Ras-response element binding factor 2 (RBF-2), comprised of Upstream Stimulatory Factor 1 and 2 (USF1/2) and TFII-I (GTF2I) (Estable et al, 1999) (Malcolm et al, 2007). Mutation of the RBE1 and RBE3 elements inhibit induction of HIV-1 in response to T cell activation (Chen et al, 2005) (Malcolm et al, 2007) (Malcolm et al, 2008), although the mechanism for this effect has not been elucidated. Here, we demonstrate that the TFII-I component of RBF-2 interacts with and recruits TRIM24 the HIV-1 LTR. TRIM24 promotes transcriptional elongation from the viral promoter in response to T cell signaling through enhanced recruitment of CDK9. Furthermore, TRIM24 and TFII-I co-occupy a significant proportion of loci genome-wide, and regulate numerous genes involved in cellular adhesion for T cell activation response. These observations provide mechanistic insight into the function of TFII-I and regulation of HIV-1 transcription for control of reactivation from provirus latency and reveal a novel mechanism for TRIM24 as a transcriptional coactivator for proviral transcriptional elongation. 2.2 Results 2.2.1 TFII-I is required for reactivation of HIV-1 provirus Mutation of the RBE3/1 binding sites for RBF-2/ TFII-I prevents induction of HIV-1 in response to T cell signaling (Chen et al, 2005) (Dahabieh et al, 2011). These cis-elements are also necessary for binding of USF1/2, and YY1 (Bernhard et al, 2013), and so to examine the effect of TFII-I in isolation on LTR-directed transcription we knocked down its expression using shRNA (Fig. 2.1B) in the Jurkat Tat mHIV-Luciferase line where luciferase is expressed from the HIV-1 5' LTR as a fusion with p24gag (Fig. 2.1A) (Bernhard et al, 2011). We observed that knockdown of TFII-I impaired expression of luciferase in these cells treated with PMA (Fig. 2.1C), or a combination of PMA and ionomycin, which activate signaling pathways stimulated by T cell activation (Fig. 2.1D). 23 Similar results were observed with knockdown of TFII-I by shRNA in the JLat10.6 cells (Fig. 2.1F), which bears a full-length HIV-1 provirus in which Nef is substituted for GFP (Jordan et al, 2003) (Fig. 2.1E). In the JLat10.6 cell line, we observed that TFII-I depletion inhibited reactivation of provirus expression in response to a variety of previously characterized LRAs, with the notable exception of the histone deacetylase inhibitor (HDACi) suberanilohydroxamic acid (SAHA) (Fig. 2.1G). In addition, we examined whether T-cell activation affected expression of TFII-I or TRIM24 and found that PMA/ionomycin treatment did not alter protein levels (Fig. S2.1A) although a slight increase in GTF2I (TFII-I) mRNA abundance was detected (Fig. S2.1B), but no effect on TRIM24 mRNA (Fig. S2.1C). Consequently, activation of HIV-1 transcription in response to T-cell signaling likely does not involve the elevated expression of TFII-I or TRIM24 protein Collectively, these results support previous observations showing that mutation of the RBE cis-elements, which bind TFII-I, impairs response of the 5' LTR to T cell activation signals (Chen et al, 2005) (Malcolm et al, 2007) (Malcolm et al, 2008) (Dahabieh et al, 2011). 24 Figure 2.1 TFII-I promotes HIV-1 expression. A: Schematic representation of the reporter virus integrated into the Jurkat Tat mHIV-Luciferase cell line, where luciferase is expressed from the 5' LTR as a fusion with p24gag. B: Jurkat mHIV-Luciferase cells were transduced with pLKO shRNA vector control (lane 1) or TFII-I shRNA expressing lentivirus (lane 2). Immunoblots were performed on whole cell lysates prepared 8 days post puromycin selection, with antibodies against TFII-I or Tubulin. C: Jurkat mHIV-Luciferase cells transduced with control shRNA or TFII-I shRNA expression vectors were left untreated (Ve, DMSO) or stimulated with 20 nM PMA for 4 hours prior to measuring luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. D: Transduced Jurkat mHIV-Luciferase cells were treated with 20 nM PMA/ 1 µM ionomycin for the indicated time prior to measuring luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. E: Schematic representation of the Jurkat based JLat10.6 HIV-1 proviral cell model, where Nef is replaced with GFP and the virus is rendered replication incompetent by a mutation within Env. F: JLat10.6 cells were transduced with empty Control shRNA vector (lane 1) or TFII-I targeting shRNA (lane 2). Following selection with puromycin for 4 days, whole cell lysates were collected and analyzed by western blot using antibodies against TFII-I or Tubulin. G: 4 days post-shRNA infection, transduced JLat10.6 cells were treated with DMSO (Ve), 1 µM ionomycin, 10 ng/µL TNFα, 10 µM JQ1, 10 µM SAHA, 10 nM PEP005, or 10 nM PMA. Following 20 hr incubation, cells were analyzed by flow cytometry (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 2.2.2 TFII-I interacts with TRIM24 To characterize mechanism(s) for regulation of HIV-1 transcription by TFII-I, we looked for novel interacting proteins using a 2-hybrid screen modified for use with transcriptional activator proteins (Hirst et al, 2001). Using a GAL4-TFII-I bait protein we isolated multiple clones encoding TUP1-TRIM24 fusions from a human T cell cDNA library. To determine whether this interaction identified in yeast can be detected in human cells, we co-transfected HEK293T cells with plasmids expressing epitope tagged TFII-I and TRIM24, where we observe TRIM24-Myc associated with TFII-I-Flag immunoprecipitated from cell lysates (Fig. 2.2A, lane 6). We performed a similar experiment in the Jurkat T cell line where we expressed TRIM24-Flag and observed interaction with endogenous TFII-I by co-immunoprecipitation (Fig. 2.2B, lane 4). We also examined interaction of TFII-I and TRIM24 in HEK293T cells using BioID proximity labeling (Fig. 2.2C-E) (Branon et al, 2018), where we transfected HEK293T cells with plasmids expressing TFII-I or GFP as a fusion with BirA* (TurboID) and Myc epitope tags (Fig. S2.2A). Upon biotin addition, we observed that the TFII-I and GFP TurboID fusions cause biotinylation of cellular proteins within 1 hour, and this effect persisted up to 24 hours (Fig. S2.2B). Importantly, we find that TRIM24 becomes biotinylated in cells expressing the TFII-I-25 BirA* fusion (Fig. 2.2E, lane 6, asterisk), but not in cells expressing GFP-BirA* (Fig. 2.2E, lane 5). Proximity labeling of TRIM24 by TFII-I-BirA* indicates that these factors associate in the context of living cells and supports the contention that TFII-I and TRIM24 interact in vivo. Figure 2.2 TRIM24 interacts with TFII-I. A: HEK293T cells were transfected with an empty vector or plasmids expressing TFII-I-Flag or TRIM24-Myc (Lanes 1 to 4). Lysates were analyzed by immunoblotting with antibodies against the Flag, Myc, or Tubulin as indicated. Cells co-expressing TFII-I-Flag and TRIM24-Myc were immunoprecipitated with control (IgG) (lane 5) or Flag (lane 6) antibodies and complexes were analyzed by immunoblotting with Flag and Myc antibodies as indicated. B: Lysates from Jurkat cells bearing a vector control (EV, lane 1) or a TRIM24-Flag expression vector (lane 2) were analyzed by immunoblotting with antibodies against Flag or TFII-I. Jurkat cells expressing TRIM24-Flag were immunoprecipitated with control (IgG, lane 3), or anti-Flag (lane 4) antibodies, and complexes were analyzed by immunoblotting with anti-Flag or anti-TFII-I antibodies as indicated. C, D, E: 26 HEK293T (lane 1) or cells expressing GFP-TurboID-Myc (lane 3 and 5), TFII-I-TurboID-Myc (lane 4 and 6), or an empty vector (lane 2) were incubated with 500 µM biotin for 1 hr. Cell lysates (lanes 1-4) or TRIM24 immunoprecipitants (lanes 5-6) were analyzed by immunoblotting with antibodies against Myc (C), TRIM24 (D), or with Streptavidin-HRP (E). Biotinylated TRIM24 is indicated with a white asterisk (E, lane 6, ~130 kDa). 2.2.3 TRIM24 activates expression from the HIV-1 LTR Because TRIM24 interacts with TFII-I in both HEK293T and Jurkat T cells, we examined whether this factor affected expression from the HIV-1 LTR. We co-transfected HEK293T cells with an LTR-luciferase reporter construct and increasing amounts of plasmid expressing TRIM24-Myc or TFII-I-Flag (Fig. 2.3A). We observed a dose dependent, three-fold increase in transcriptional activity of LTR-Luc relative to endogenous levels of TRIM24 (Fig. 2.3B). In contrast, we observed only a modest 1.7 fold increase in transcription when TFII-I-Flag was overexpressed. These data are consistent with the previously described role of TFII-I at the HIV-1 LTR (Chen et al, 2005) (Dahabieh et al, 2011), and suggests that TRIM24 represents a required co-factor for transcriptional activation. To further examine the role of TRIM24 for activation of HIV-1 expression, we examined the effects of TRIM24 and TFII-I knockdown. We first validated siRNA SmartPools against TRIM24 and TFII-I in transiently transfected HEK293T cells. Both pools were efficient at knocking down expression as determined by immunoblotting, within 48 hours post transfection; however, knockdown was more pronounced at 72 hours (Fig. 2.3C). We then co-transfected siRNA pools with an LTR-luciferase reporter construct into HEK293T cells, and measured luciferase activity 72 hours later. Here we observed a 40% decrease in luciferase activity in TRIM24 knockdown cells, relative to the control (Fig. 2.3D). Consistent with results from overexpression, knockdown of TFII-I resulted in a more modest 25% decrease in luciferase activity, and knockdown of both TFII-I and TRIM24 produced an effect similar to knockdown of TRIM24 alone (Fig. 2.3D). We also assessed the effect of TRIM24 for Tat-dependent activation of the HIV-1 LTR. To this end, we transfected HEK293T cells with LTR-luciferase reporter constructs, either bearing or lacking the Tat-responsive element TAR, in the presence or absence of a plasmid expressing the viral transactivator Tat, as previously described (Dahabieh et al, 2011). Regardless of the presence of TAR and/or Tat, overexpression of TRIM24 and TFII-I resulted in elevated expression of luciferase (Fig. 2.3E), indicating that the effect of TRIM24 on LTR-directed expression is largely independent of Tat function. Finally, we analyzed the effect of TFII-I or TRIM24 overexpression on the induction of an LTR construct bearing RBE3/1 elements with mutations known to abrogate RBF-2 (TFII-I) binding (Dahabieh et al, 2011). We found that TFII-I-Flag transfection did not induce luciferase 27 expression from an HIV-1 LTR reporter in which the RBE elements were mutated (Fig. 2.3F). Upon transfection of TRIM24-Myc, we observed a slight increase in luciferase expression from the mRBE3/1 LTR reporter, however, the observed luciferase induction was far less than for that of the wildtype LTR (Fig. 2.3F). These results confirm that TFII-I and TRIM24 require intact RBE3 and RBE1 elements for full induction of HIV-1 LTR transcription. Figure 2.3 HIV-1 LTR transcription is activated by TRIM24. A: HEK293T cells were transfected with plasmids expressing TFII-I-Flag (lane 2), TRIM24-Myc (lane 3), or an empty vector (EV, lane 1). Lysates were analyzed by immunoblotting against Myc, Flag, or Tubulin as indicated. B: HEK293T cells co-transfected with an LTR-Luciferase reporter plasmid and TFII-I-Flag or TRIM24-Myc expression vectors were analyzed for luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: HEK293T cells were transfected with siRNA against TFII-I or TRIM24 (lanes 3-4 and lanes 7-8). Lysates were collected 2- (lanes 1-4) and 3-days (lanes 5-8) post transfection and analyzed by immunoblotting with anti-TRIM24, anti-TFII-I, or anti-GAPDH antibodies as indicated. Loading control was performed on individual gel. D: HEK293T cells co-transfected with an LTR-Luciferase reporter plasmid and siRNA targeting TFII-I and/or TRIM24 were analyzed for luciferase expression (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. E: Luciferase assay was 28 performed using HEK293T cells co-transfected with an LTR-Luciferase reporter plasmid and TRIM24-Myc and/or TFII-I-Flag expression vector, in the presence (+ Tat) or absence (-Tat) of Tat expression vector. The LTR-Luciferase reporter contained (+TAR) or lacked the TAR (-TAR) stem loop region (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. F: Wildtype LTR-Luciferase reporter or LTR-Luciferase reporter with mutated RBE3 and RBE1 elements (mRBE3/1) were co-transfected with an empty vector plasmid (EV) or plasmids expressing TFII-I-Flag or TRIM24-Myc (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 2.2.4 TRIM24 is required for induction of HIV-1 provirus expression The results shown above indicate that TRIM24 interacts with TFII-I, and its presence correlates with activation of HIV-1 LTR-directed transcription in HEK293T cells. We next examined the requirement of this factor for HIV-1 provirus expression in T cells. We found that T cell activation in response to PMA/ ionomycin treatment did not alter TRIM24 expression (Fig. S2.1A, S2.1C). We used shRNA to knockdown TRIM24 expression (Fig. 2.4A) in the Jurkat mHIV-Luciferase cell line where luciferase expression is driven by the 5’ LTR (Fig. 2.1A). For all shRNAs used, we observed a substantial decrease in luciferase expression in response to PMA compared to the non-targeting control shRNA vector (Fig. 2.4B). Similarly, we performed TRIM24 shRNA knockdown (Fig. 2.4C) in JLat10.6 cells and observed that TRIM24 depletion inhibited latency reversal in response to all agonists and latency reversing agents used, as measured by the percent of cells that expressed GFP (Fig. 2.4D). We also examined the effect of TRIM24 on HIV-1 expression using CRISPR-Cas9 mediated gene disruption (Fig. S2.3A) in the Jurkat mHIV-Luciferase cell line. Consistent with the results with shRNAs, each TRIM24 knockout (TRIM24 KO) clonal cell line displayed significant reduction in HIV-1 expression in response to treatment with PMA, as measured by luciferase activity (Fig. S2.3B). Furthermore, induction of HIV-1 expression in response to treatment with various latency reversing agents (LRAs) was significantly inhibited in the TRIM24 knockout lines (Fig. 2.4E). Additionally, we observed that TRIM24 KO cells produced significantly impaired HIV-1 reactivation in response to T cell activation as induced by CD3/CD28 mediated T cell receptor cross linking (Fig. 2.4F). To confirm that effects on HIV-1 expression observed in the TRIM24 knockout cells is not caused by an indirect effect, we generated a Jurkat Tat T cell line in which endogenous TRIM24 is deleted, and the factor is complimented by conditional expression of TRIM24 under control of a tetracycline repressed promoter (Fig. 2.5A). The TRIM24 Tet-Off cell line was infected with a modified dual HIV-1 reporter derivative (Red-Blue HIV, RBH, Fig. 2.5B) which expresses BFP from the 5' LTR and mCherry from an internal CMV promoter to enable detection of infected cells independently from LTR-directed transcription (Dahabieh et al, 2014). Three days post infection with RBH we depleted TRIM24 by addition of doxycycline, and cells 29 were left untreated, or stimulated with PMA and ionomycin; the infected cells were analyzed for latently and productively infected cells the following day by flow cytometry (Fig. 2.5B). In this analysis we found that depletion of TRIM24 caused a dramatic reduction in the proportion of productively infected cells, as indicated by cells expressing CMV-mCherry and BFP from the LTR, in both the untreated (Fig. 2.5C, Un - Dox) and PMA/ ionomycin treated (Fig. 2.5C, PMA/Ion - Dox) populations compared to cells expressing TRIM24 (Fig. 2.5C, Un - Ve, and PMA/Ion - Ve). In untreated cells we found that TRIM24 depletion caused near complete production of latent provirus (Fig. 2.5D, Un), and in PMA/ Ionomycin treated cells we observed a reduction in productively infected cells to ~10% from the normal proportion of ~50% (Fig. 2.5D, PMA/Ion). Furthermore, in both untreated and PMA/ ionomycin treated cells we observed a significant reduction in levels of 5' LTR-BFP expression as determined by mean fluorescence intensity (MFI) in these populations (Fig. 2.5E). Collectively, these results indicate that TRIM24 is essential for full induction of HIV-1 expression in response to T cell activation signals as well as many LRAs. Figure 2.4 TRIM24 is an activator of HIV-1 expression in T cells. A: Jurkat mHIV-Luciferase cells were transduced with pLKO empty vector control (lane 1) or various TRIM24 targeting shRNA (lanes 2-4). Following 3 days puromycin selection, lysates from transduced cells were analyzed by immunoblotting with antibodies against TRIM24 or Tubulin. B: Jurkat mHIV-Luciferase cells transduced with control shRNA vector or TRIM24 targeting shRNA were left untreated (Ve, DMSO) or stimulated with 20 nM PMA for 4 hours prior to measuring luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: JLat10.6 cells were transduced with a Control shRNA vector or a TRIM24 targeting shRNA. Following puromycin selection, whole cell lysates were extracted and subject to immunoblotting 30 against TRIM24 or Tubulin. D: Control shRNA or TRIM24 targeting shRNA transduced JLat10.6 cells were cultured for 4 days with puromycin and subsequently incubated with DMSO (Ve), 1 µM ionomycin, 10 µM JQ1, 10 µM SAHA, 10 nM PEP005, and 5 nM or 10 nM PMA, cells were examined 20 hr later by flow cytometry (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. E: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were incubated with DMSO (Ve), 1 µM ionomycin, 10 ng/µL TNFα, 10 µM JQ1, 10 µM SAHA, 10 nM PEP005, 10 nM PMA or 10 nM PMA/ 1 µM ionomycin for 4 hrs prior to measuring luciferase expression (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. F: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase were cultured in the presence of CD3/CD28 coated beads with luciferase expression analyzed at the indicated time points (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. Figure 2.5 TRIM24 knockout encourages establishment of immediate latency. A: Wildtype, TRIM24 KO, or TRIM24 TetOff Jurkat cells were untreated or incubated with 1 µg/mL doxycycline for 20 hr, when whole cell lysates were analyzed by immunoblotting with TRIM24 or Tubulin antibodies. B: Schematic depiction of the Red-Blue-HIV-1 reporter virus (RBH) assay. TRIM24 knockout Jurkat cells transduced with a TetOff TRIM24 expression lentivirus were infected with RBH. 3 days post-infection, cells were untreated or incubated with 1 µg/mL doxycycline (DOX); 2hr post DOX treatment, cells were treated with a vehicle control (DMSO) or 10 nM PMA/ 1 µM ionomycin. Flow cytometry was performed 20 hr later to measure 31 infection (Red) and HIV-1 (Blue) reporter expression. C: Representative flow cytometric scatterplots of cells treated as described in B; mCherry intensity is displayed on the Y-axis, and BFP expression on the X-axis. For each scatterplot, the bottom left quadrant indicates uninfected cells (mCherry-/BFP-), the top left indicates the latent population(mCherry+/BFP-), the top right indicates productively infected cells (mCherry+/BFP+), and the bottom right depicts noise generated from viral rearrangements (BFP+). D: Summary of scatterplot data produced from RBH TRIM24 rescue experiments. Shown is the percentage of productively infected cells, with error bars representing standard deviation (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. E: Same as D, but data is presented as the change in BFP mean fluorescent intensity (MFI) as compared to RBH uninfected, fluorescent negative cells (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 2.2.5 Interaction of TRIM24 with the HIV-1 LTR requires TFII-I To further assess the relationship between TFII-I and TRIM24 for regulation of HIV-1 expression, we examined their occupancy on the HIV-1 LTR using ChIP-qPCR. Consistent with previous observations (Bernhard et al, 2013), we observe interaction of TFII-I at the LTR regions representing both RBE3 and RBE1 in the presence or absence of PMA stimulation (Fig. 2.6A), and a similar result was observed for TRIM24 (Fig. 2.6B); however we do observe recruitment of NFκB p65 to the enhancer region in response to PMA treatment of these same cells (Fig. S2.4). These observations indicate that, consistent with the interaction as measured by co-IP and BioID (Fig. 2.2), TRIM24 co-localizes with TFII-I on the HIV-1 LTR in human T cells. We similarly observed association of TFII-I and TRIM24 with the HIV-1 LTR in the HeLa-derived TZM-bl cell line, in both unstimulated and PMA/ ionomycin treated cells (Fig. S2.5). To examine the requirement of TFII-I for recruitment of TRIM24 to the LTR and vice versa, we performed ChIP-qPCR in cells where these factors were depleted. We first confirmed that depletion of TFII-I and TRIM24 resulted in the loss of their respective ChIP signals as measured with primers specific for the HIV-1 LTR (Fig. 2.6C, 2.6D). We found that association of TFII-I with the LTR was unaffected by TRIM24 KO (Fig. 2.6E). In contrast, interaction of TRIM24 with both the RBE3 and RBE1 regions was significantly reduced in cells where TFII-I expression was knocked down with shRNA (Fig. 2.6F). Thus, loss of TRIM24 has no effect on binding of TFII-I to its sites on the LTR but interaction of TRIM24 with the LTR is dependent upon TFII-I. These observations support the contention that TFII-I directly recruits TRIM24 to the HIV-1 LTR. 32 Figure 2.6 TFII-I/ TRIM24 co-localize to the HIV-1 LTR. A, B: Jurkat Tat mdHIV Clone 11 cells were left untreated (Ve, DMSO) or incubated with 50 nM PMA for 24 hrs. ChIP-qPCR was performed using anti-TFII-I (A) or anti-TRIM24 (B) antibodies. C: ChIP-qPCR analysis was performed using TFII-I antibodies with Jurkat mHIV-Luciferase cells transduced with pLKO lentiviral control shRNA or TFII-I targeting shRNA. D: As in C but anti-TRIM24 antibodies were used for ChIP analysis. E: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were subject to ChIP-qPCR analysis using anti-TRIM24 antibodies. F: As in E but anti-TFII-I antibodies were used for ChIP analysis. All ChIP-qPCR results are normalized by subtraction of values produced with sample paired non-specific IgG (n = 2 - 4, mean ± SD). Statistical analysis was performed using unpaired t-test. 2.2.6 TRIM24 stimulates HIV-1 transcriptional elongation We examined the mechanism for regulation of HIV-1 transcription by TRIM24 by measuring the effect of this factor for initiation and elongation of RNAP from the LTR promoter. For this we employed an RT-PCR assay for mRNAs representing initiating versus elongating 33 (Proximal, Gag) transcripts (Fig. 2.7A) (Zhu et al, 2012) (Yukl et al, 2018). In these experiments, we observed a small increase in abundance of HIV-1 RNA associated with paused RNA Polymerase II upon TRIM24 depletion as compared to wildtype in untreated cells (Fig. 2.7B, Initiation). In contrast, we observed a marked decrease in both proximal and Gag elongating mRNA transcripts in TRIM24 KO cells relative to wildtype untreated cells (Fig. 2.7B). Similarly, in cells stimulated with PMA, we observe significant reduction in promoter proximal and gag elongated transcripts, and to a lesser extent, initiation transcripts, in TRIM24 KO cells relative to WT (Fig. 2.7C), indicating that loss of TRIM24 function causes a defect in transcriptional elongation from the HIV-1 LTR. We note that this assay is complicated by the fact that all HIV-1 transcripts possess the same first 69 nucleotides of viral mRNA (Fig. 2.7A) and consequently the difference in initiation transcripts between TRIM24 KO and wildtype cells is exaggerated in PMA treated cells because of accumulation of elongated mRNAs. To further examine the effect of TRIM24 on HIV-1 transcription, we measured its role for recruitment and modification of RNAP. We observed enhanced interaction of RNAP with the LTR in cells treated with PMA and ionomycin, relative to untreated cells (Fig. 2.7D, WT). In TRIM24 KO cells we observed a slight increase in RNA Pol II occupancy at the LTR promoter in untreated cells, but did not observe a significant difference in association of RNA Pol II with the LTR in cells treated with PMA and ionomycin (Fig. 2.7D, TRIM24 KO) compared to WT cells, indicating that TRIM24 is dispensable for initial recruitment of RNAPII to the LTR promoter. RNA Polymerase II becomes phosphorylated at S2 on the CTD during initiation of transcription, an effect that promotes transcriptional elongation (Buratowski, 2009). As expected, ChIP-qPCR analysis of phosphorylated Ser2 (pS2) CTD showed enrichment on the LTR upon stimulation of T cell signaling in wildtype cells (Fig. 2.7E). However, accumulation of pS2 modified RNAPII was significantly reduced in the TRIM24 KO cell line (Fig. 2.7E). Phosphorylation of CTD S2 is produced by CDK9 of P-TEFb (Bieniasz et al, 1999), and consequently we also examined whether the TRIM24 KO affected recruitment of CDK9 to the LTR. In agreement with observations that P-TEFb is sequestered by the 7SK snRNP complex in unstimulated T cells (Mbonye et al, 2013), ChIP-qPCR using antibodies against CDK9 revealed low levels of association at the LTR in untreated cells, but significant enhancement in wildtype cells treated with PMA and ionomycin (Fig. 2.7F). In contrast, we did not observe a significant increase of CDK9 occupancy in stimulated TRIM24 KO cells (Fig. 2.7F). Importantly, we do not observe an effect of the TRIM24 knockout on expression of CDK9 or cyclin T1 proteins (Fig. S2.6). Consistent with the above results, and previous observations (Mbonye et al, 2018), we find that treatment of cells with a CDK9 inhibitor prevented reactivation of latent HIV-1 in response to treatment with PMA (Fig. S2.7). Collectively, these findings indicate that TRIM24 mediates activation of HIV-1 expression by promoting recruitment of CDK9 to the viral promoter and facilitating the transition of RNAPII into an actively elongating complex. 34 Figure 2.7 TRIM24 stimulates elongation of HIV-1 transcription. A: Schematic representation of the 5' LTR TAR and Gag-encoding regions, indicating primer pairs used to measure Initiation (+10-59 bp), Proximal (+29-180 bp), or Gag (+456-612 bp) HIV-1 transcript abundance. B, C: Wildtype and TRIM24 KO Jurkat mHIV-Luciferase cells were cultured under normal conditions (B) or with 20 nM PMA for 4 hrs (C). RNA was extracted, cDNA synthesized, and RT-PCR was performed using primers to detect Initiation, Proximal and Gag RNA sequences with data normalized to the untreated wildtype control (n = 3, mean ± SD). Statistical analysis was performed using unpaired T-test. D, E, F: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were left untreated or stimulated with 20 nM PMA/ 1 M ionomycin for 4 hrs. ChIP-qPCR analysis was performed using anti-RNAPII (D), anti-RNAPII pS2 (E), or anti-CDK9 (F) antibodies. Results were normalized by subtraction of values 35 produced with sample paired non-specific IgG (n = 3 - 7, mean ± SD). Statistical analysis was performed using two-way ANOVA. 2.2.7 Loss of TRIM24 does not cause substantial effects on LTR-associated histone modification Upon integration into the host chromosome, the HIV-1 LTR becomes associated with two phased nucleosomes termed Nuc-0 and Nuc-1, positioned at -140 and the transcriptional start site (Verdin, 1991) (el Kharroubi & Verdin, 1994). These nucleosomes modified with H3K27ac is associated with activated transcription (Tripathy et al, 2015), whereas the H3K9me3 modification is associated with transcriptional repression (Bernhard et al, 2011) (Imai et al, 2010). We examined the effect of TRIM24 knockout on these modifications at the LTR and observed only a slight increase in H3K27ac in TRIM24 KO cells as compared to wildtype (Fig. 2.8A), and a minor decrease in H3K9me3 (Fig. 2.8B). These results indicate that loss of TRIM24 function does not cause significant alterations in chromatin modification at the LTR and indicate its effect on HIV-1 transcription specifically involves a role in promoting transcriptional elongation. Figure 2.8 Loss of TRIM24 does not induce restrictive LTR chromatin organization. A, B: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were subject to ChIP-qPCR using antibodies targeting acetylated H3K27 (A) or trimethylated H3K9 (B). ChIP-qPCR results were normalized by subtraction of values produced with sample paired non-specific IgG (n = 2 - 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 2.2.8 Regulation of HIV-1 by TRIM24 is independent of TRIM28 TRIM24 (TIF1α) along with the structurally similar TRIM28 (KAP1, TIF1β), TRIM33 (TIF1γ), and TRIM66 (TIF1δ) comprise the Transcription Intermediary Factor (TIF) subfamily of the Tripartite Motif (TRIM) family of proteins (Hatakeyama, 2017) (Peng et al, 2000). These proteins form regulatory complexes; in mice, TRIM24, TRIM28, and TRIM33 associate to suppress the development of hepatocellular carcinoma (Herquel et al, 2011), while human 36 TRIM28 interacts with TRIM24 to promote prostate cancer tumorigenesis (Fong et al, 2018). Of note, TRIM28 was implicated in regulation of HIV-1 latency by modification of CDK9 (Ma et al, 2019), but overall the role of TRIM28 for regulation of HIV-1 transcription is contentious (Ait-Ammar et al, 2021) (Krasnopolsky et al, 2020) (McNamara et al, 2016) (Morton et al, 2019) (Taura et al, 2019). Because of these observations we examined whether association of TRIM28 with the HIV-1 LTR was dependent upon TRIM24. In this analysis we did not observe a difference in association of TRIM28 with the HIV-1 LTR in TRIM24 KO cells compared to WT (Fig. S2.8A). Furthermore, the TRIM24 knockout did not alter expression of TRIM28 mRNA (Fig. S2.8B) or protein (Fig. S2.8C). These observations suggest that the effect of TRIM24 on regulation of HIV-1 expression is independent of TRIM28. 2.2.9 TFII-I and TRIM24 co-regulate specific subsets of cellular genes Having discovered that TFII-I recruits TRIM24 to the HIV-1 LTR to promote transcriptional elongation, we next examined the extent that these factors contribute to co-regulation of cellular genes. We first examined publicly available ENCODE TFII-I and TRIM24 ChIP-seq datasets derived from the K562 lymphoblast cell line (ENCODE Project Consortium, 2012) (Luo et al, 2020). From these we identified genomic binding sites of TRIM24 and TFII-I, for which the latter displayed notably higher coverage (Fig. S2.9A, S2.9B), and comparison of these revealed that just under 10% of TRIM24 bound genomic loci were co-occupied with TFII-I (Fig. S2.9A). Analysis of binding sites in relation to the nearest gene revealed a predilection for introns, intergenic, and promoter regions for both TFII-I and TRIM24 (Fig. S2.9C). These observations are consistent with previous ChIP-seq experiments performed in non-K562 cell lines (Bayarsaihan et al, 2012) (Fong et al, 2018). Strikingly, analysis of sites co-occupied by TFII-I and TRIM24 resulted in enriched preference for promoters (Fig. S2.9C, bottom), indicating that cellular gene regulation by TFII-I/TRIM24 potentially requires proximity to transcriptional start sites. To further examine the role of TRIM24 and TFII-I for gene expression, we used RNA-Seq to identify differentially expressed genes (DEG) in Jurkat Tat T cells depleted of TRIM24 by gene knockout or TFII-I using shRNA. In this analysis we identified 823 genes that were differentially expressed upon knockout of TRIM24 (Fig. S2.10A), and 2588 genes altered upon TFII-I knockdown (Fig. S2.10B), and in agreement with their function as activators or repressors, we observed near equal distribution between up- and down-regulation of DEG (Fig. S2.10). We examined global function of TRIM24-responsive genes by performing Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis to determine enrichment of biological process gene ontology (GO) terms (Fig. 2.9). RNA-seq analysis identified DEG between wildtype and TRIM24 KO T cells treated with PMA and ionomycin (Fig. S2.10A), and consistently we observe enrichment of GO terms related to T cell activation, including regulation of signal transduction, leukocyte activation and cytokine production (Table S2.2), indicating a role for TRIM24 in T cell immune response. We also observed GO terms related to cell adhesion (Fig. 2.9A), and although Jurkat T cells grow in suspension they adhere to culture surfaces upon 37 activation of T cell signaling (Albelda et al, 1994) (Roy et al, 1998). Consistently, we observe that TRIM24 KO Jurkat cells are significantly less adherent upon treatment with PMA and ionomycin compared to wildtype cells (Fig. 2.9B). It is well established that expression of cell adhesion molecules are an important component of T cell mediated immune response (Douglass & Vale, 2005) (Dutta et al, 2017) (Monks et al, 1998). In contrast, TRIM24 KO does not abrogate global T cell activation, as the majority of T cell activation induced and repressed genes are similarly regulated in TRIM24 KO cells compared to wildtype (Fig. S2.11A-C). Because of the relationship between TFII-I and TRIM24 for regulation of HIV-1, we examined the extent that these factors regulate common cellular transcriptional programs by analysis of genes whose differential expression were similar upon loss of TRIM24 or TFII-I. We found that a significant proportion of TRIM24 KO DEG exhibited similar regulation patterns as did TFII-I knockdown, with 123 common down-regulated genes (24.9% of TRIM24 KO down-regulated genes) and 119 common up-regulated genes (36.1% of TRIM24 KO up-regulated genes) (Fig. 2.9C, Fig. S2.12). DAVID analysis of these similarly regulated genes represented many of the same GO terms as TRIM24 KO DEG, indicating that TFII-I facilitates the TRIM24 transcriptional program to some extent. Of note, genes that are dysregulated similarly with the loss of TRIM24 or TFII-I highlight cellular adhesion (Fig. 2.9D), as 37 of the common DEG are involved in adhesion with nearly half up-regulated or down-regulated (Fig. 2.9E). These 37 genes represent 31% of all genes involved in cell adhesion that are mis-regulated upon loss of TRIM24 (Fig. S2.13). Collectively, these results highlight that TFII-I augments the genome-wide TRIM24 transcriptional program, and indicates that regulation of many cellular genes may involve direct recruitment of TRIM24 by TFII-I. 38 Figure 2.9 TFII-I enhances the TRIM24 transcriptional program. A: PMA/ ionomycin treated TRIM24 KO DEG display enrichment of cell adhesion related biological processes as identified with DAVID online tool gene ontology analysis. DAVID adopts Fisher's Exact test to statistically measure gene-enrichment. B: Wildtype or TRIM24 KO Jurkat Tat T cells were left untreated or stimulated with 20 nM PMA/ 1 M ionomycin for 2 hrs. The proportion of cells that adhered to the flask culture surface or remained in suspension was determined (n = 2, mean ± SD). C: Heat map depiction of significantly down-regulated (blue) or up-regulated (red) genes that are common to TRIM24 KO and TFII-I knockdown cells (PMA/ ionomycin treated). Relative gene expression was determined by DESeq2 analysis of n = 3 RNA-seq samples. D: DAVID online tool gene ontology analysis of TRIM24 KO and TFII-I knockdown common DEG (PMA/ ionomycin treated) identified enrichment of cell adhesion related biological processes. DAVID adopts Fisher's Exact test to statistically measure gene-enrichment. E: Heat map showing significantly down-regulated (blue) or up-regulated (red) genes that are common to TRIM24 KO and TFII-I knockdown (PMA/ ionomycin treated) and are involved in cellular adhesion. n = 3 RNA-seq samples were analyzed by DESeq2 to determine relative gene expression. 39 2.3 Discussion In this study we identified an interaction between TFII-I and TRIM24, which is required for efficient transcriptional elongation from the HIV-1 LTR promoter. The important role of transcriptional elongation for regulation of HIV-1 expression was recognized since discovery that the viral transactivator Tat recruits P-TEFb to paused RNA Polymerase II complexes through interaction with nascent TAR RNA (Bieniasz et al, 1999) (Ping & Rana, 2001). Accordingly, loss of Tat in cells bearing transcriptionally silenced provirus is thought to represent a significant barrier for reactivation of virus expression (Karn, 2011) (Ott et al, 2011). One report has indicated that several commonly studied latency reversing agents (LRA), including PEP005 and Ingenol cause reactivation of viral expression by promoting transcriptional elongation and splicing of sub-genomic transcripts, rather than initiation of transcription (Yukl et al, 2018), although mechanism(s) for this effect were not determined. Our results indicate that Tat or TAR are not required for stimulation of HIV-1 transcription by TRIM24. Consequently, it is possible that the effect of TFII-I-TRIM24 for recruitment of CDK9, and phosphorylation of CTD S2, might represent a priming mechanism to kick start synthesis of elongated and spliced transcripts for production of Tat protein (Mbonye & Karn, 2014) which would produce the characteristic positive feedback loop for viral gene expression (Razooky & Weinberger, 2011) (Razooky et al, 2015) (Weinberger & Shenk, 2007). The function of TFII-I for regulation of HIV-1 transcription has been enigmatic. Binding sites for TFII-I, in conjunction with USF1 and 2 are highly conserved on LTRs from patients (Estable et al, 1996), and these elements are necessary for reactivation of HIV-1 transcription in response to T cell signaling (Chen et al, 2005). This observation is consistent with results indicating that TFII-I is involved in activation of c-Fos expression in response to MAPK signaling (Kim & Cochran, 2000), but a specific mechanistic role of TFII-I for stimulation of transcription from the HIV-1 LTR has not been identified. TFII-I interacts with multiple additional DNA binding factors on various cellular promoters, including STAT1, STAT3, SRF, ERSF, and on the HIV-1 LTR with USF1 and USF2 (Roy, 2012) (Chen et al, 2005). In the latter case, interaction of USF1/2 with the upstream RBE3 element on the HIV-1 LTR requires TFII-1 to produce a specificity unique to this complex (Malcolm et al, 2008). These observations suggest that TFII-I plays a significant role for directing cooperative interaction of various sequence specific factors with regulatory cis-elements. Our finding that TFII-I recruits the co-activator TRIM24 suggests this factor plays an additional more elaborate role for gene regulation than acting as a chaperone for DNA binding partners. It will be interesting to determine how additional functions attributed to TRIM24 are separated from those involved in interaction with TFII-I and recruitment to the HIV-1 LTR. TRIM24 was previously identified as a co-activator for nuclear receptors, including for estrogen (Thénot et al, 1997), androgen (Kikuchi et al, 2009), and androstane (Kanno et al, 2018). However, the mechanism(s) by which TRIM24 causes transcriptional activation has not been identified. Considering our results, we propose that TRIM24 may also activate 40 transcriptional elongation for nuclear receptor target genes. Our results indicate that TRIM24 enhances association of CDK9 to the HIV-1 LTR, and the simplest possibility is that TRIM24 may directly interact with CDK9 or cyclin T to promote recruitment of P-TEFb (Fig. 10). Alternatively, TRIM24 may modify additional factors at the LTR core promoter to enable access or recruitment of P-TEFb. TRIM24 has E3-ubiquitin ligase activity, and was shown to promote degradation of p53 through direct ubiquitylation (Allton et al, 2009) (Jain et al, 2014). TRIM24 was also found to modify the functions of CBP and TRAF3 by catalyzing K63-linked ubiquitination. TRIM24 mediated K63 ubiquitination of CBP inhibited macrophage polarization (Yu et al, 2019), while this modification of TRAF3 was also observed as part of the anti-viral response to VSV infection (Zhu et al, 2020). Elucidation of the precise role of TRIM24 as a transcriptional co-activator will require a detailed understanding of its interactions with the general transcription factor machinery, and our results provide a novel glimpse towards understanding the function of this factor regulation of gene expression. It is believed that TRIM24 contributes to cancer progression by operating as a coactivator for nuclear hormone receptors (Groner et al, 2016) (Tsai et al, 2010). However, the majority of chromatin-associated TRIM24 is not co-localized with estrogen receptor (ER) in breast cancer tissue, and overexpression of TRIM24 correlates with poor patient survival independent of ER status (Tsai et al, 2010). Here, we found that TFII-I and TRIM24 display a substantial degree of genome-wide co-localization and that loss of TRIM24 alters the immune response, displayed in part by decreased cellular adhesion. A role for TRIM24 in immune function has previously been identified in that T-helper 2 cells deficient of TRIM24 are refractory to house dust mite-induced allergic inflammatory activation, although a mechanism for this effect was not determined (Perez-Lloret et al, 2016). Furthermore, MCF7 breast cancer cells deficient in TRIM24 also display a reduced cell adhesion phenotype that is thought to contribute to cancer cell proliferation, migration, and metastasis (Appikonda et al, 2018). Consequently, we believe it unlikely that TRIM24-regulated transcriptional programs such as cancer and inflammation are the sole result of nuclear receptor co-activator function, and that gene regulation by TRIM24 occurs to some degree through direct recruitment by additional DNA binding factors, including TFII-I. There is currently considerable interest in development of therapies that could be applied in addition to, or instead of, ART to eliminate latently infected cells from patients. One potential strategy, broadly termed "block and lock" is based on the rationale that preventing stochastic basal transcriptional noise of the latent provirus may discourage maintenance of the latently infected cell population (Moranguinho & Valente, 2020) (Sadowski & Hashemi, 2019). Based on results presented here, we suggest that TRIM24, and specifically the interaction between TRIM24 and TFII-I may represent an important specific target for this strategy. 41 Figure 2.10 TRIM24 is an RBF-2 cofactor that stimulates transcriptional elongation of HIV-1. The TFII-I component of RBF-2 directly interacts with, and recruits TRIM24 to the HIV-1 LTR. TRIM24 is essential for T cell signal-induced activation of chromosomally integrated provirus and is required for recruitment of P-TEFb/CDK9 to the LTR, and phosphorylation of Ser-2 of the RNA Pol II CTD, to promote transcriptional elongation. 2.4 Materials and methods 2.4.1 Cell and virus culture Jurkat E6-1, Jurkat Tat, TZM-bl, and HEK293T cells were cultured under standard conditions, 37oC and 5% CO2. Jurkat cells were cultured in RPMI-1640 media while TZM-bl and HEK293T were cultured in DMEM. All media was supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL). Vesicular stomatitis virus G (VSV-G) pseudotyped viral stocks were produced by co-transfecting HEK293T cells with a combination of viral molecular clone, psPAX, and VSV-G at a ratio of 8 μg : 4 μg : 2 μg. Transfections were performed with polyethylenimine (PEI) at a ratio of 6:1 PEI:DNA in Gibco Opti-MEMtm. Lentiviral infections were performed by plating 1x106 cells in 24-well plates with media containing 8 μg/mL polybrene and the amount of viral supernatant to give the desired multiplicity of infection (M.O.I.) as indicated. Plates were subsequently spinoculated for 1.5 hrs at 1500 rpm. 2.4.2 shRNA knockdown Jurkat mHIV-Luciferase or JLat10.6 cells were infected with pLKO empty vector or pLKO shRNA expressing lentivirus at a M.O.I. ~10. For Jurkat mHIV-Luciferase cells, TFII-I shRNA infected cells were cultured for 8 days with 7.5 µg/mL puromycin while TRIM24 42 shRNA infected cells were cultured 3 days with 3 µg/mL puromycin. For JLat10.6 cells, all shRNA infected cells were cultured 4 days with 1 µg/mL puromycin. Pools of puromycin selected cells were prepared for the indicated analysis. MISSION shRNA clones (Sigma) used for knockdown in Jurkat mHIV-Luciferase cells were as follows: TFII-I, TRCN0000019315; TRIM24-1, TRCN0000021263; TRIM24-2, TRCN0000021262; TRIM24-3, TRCN0000021259. MISSION shRNA clones (Sigma) that were used for knockdown in JLat10.6 cells were: TFII-I, TRCN0000364550; TRIM24, TRCN0000021262. 2.4.3 TRIM24 knockout TRIM24 KO cell lines were generated using CRISPR-Cas9 in the Jurkat Tat mHIV-Luciferase cell line that possesses a chromosomally integrated HIV-1 mini-virus where luciferase is expressed from the 5' LTR (Fig. 2.1A). Briefly, 2x106 cells were co-transfected with Cas9 (pU6_CBh-Cas9-T2A-BFP: Addgene #64323) and gRNA (pSPgRNA: Addgene #47108) sequences that target genomic TRIM24, using the Neon Transfection System (Invitrogen) as per the manufacturer’s instructions with the following settings: Voltage, 1350 V; Width, 20 ms; Pulse number, 3x. Knockout cells were isolated by live fluorescent activated cell sorting (Astrios Flow Cytometer) BFP positive cells into 96-well plates containing complete RPMI-1640. Clones were expanded, and knockout of TRIM24 was validated by PCR genotyping and western blotting. TRIM24 gRNA target sequences were CTGCATATTATTTAAGCAAC and GAACGAGGCCGAGAGTCGGC. 2.4.4 Immunoblotting and immunoprecipitation Western blotting was performed as previously described (Hashemi et al, 2018). Unprocessed film is included in Fig. S2.15. Antibodies were as follows: Tubulin (1:40000) - Abcam ab7291, TFII-I (1:20000) - Abcam ab134133, Flag (1:20000) - Sigma Aldrich F3165, Myc (1:2500) - Santa Cruz sc-40, TRIM24 (1:4000) - Proteintech 14208-1-AP, KAP1 (1:2500) - Proteintech 15202-1-AP, GAPDH (1:10000) - Abcam ab9484, CDK9 (1:4000) - Abcam ab239364, Cyclin T1 (1:500) - Santa Cruz sc-10750, Streptavidin-HRP (1:40000) - Abcam ab7403, Goat Anti-Rabbit-HRP (1:2000000) - Abcam ab6721, Goat Anti-Mouse-HRP (1:20000) - Pierce #1858413. Immunoprecipitations (IPs) were performed using HEK293T or Jurkat E6-1 cells. 8.33x105 HEK293T cells were plated with 2 mL DMEM in 6-well plates. The following day, cells were transfected with 4 g of the indicated construct, using 3 µg PEI per 1 µg plasmid DNA and harvested 24-48 hrs post-transfection. For Jurkat IPs, cells were transduced with TRIM24-Flag or empty vector lentivirus and experiments were performed following puromycin selection. Cells were collected, washed with ice cold PBS, suspended in DR Buffer A (10 mM HEPES-KOH pH = 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1x PIC, 0.5 mM PMSF) and incubated at 40C for 15 min. Samples were centrifuged, the cytoplasmic supernatant was discarded while the pelleted nuclei were suspended in DR Buffer C (20 mM HEPES-KOH pH = 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 1x PIC, 0.5 mM 43 PMSF) and briefly sonicated (Covaris S220 Focused-ultrasonicator). Samples were cleared by centrifugation and protein concentrations were determined using a Bradford assay (BioRad). 250 g of nuclear extract was diluted with Flag-IP Buffer (50 mM Tris-HCl pH = 8.0, 90 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1x PIC) and 2 μg of antibody was added (Flag - Sigma Aldrich F3165, mouse IgG – Santa Cruz sc-2025); the antibody – lysate mixture was incubated at 4oC with rotation for 1 hr prior to the addition of protein A/G agarose beads (Millipore, 50 L/IP) and continued overnight incubation. The samples were washed 3x with Flag-IP Buffer and eluted in 4x SDS Sample Buffer with boiling. Eluted proteins were subject to analysis by immunoblotting. 2.4.5 Chromatin immunoprecipitation and quantitative PCR Exponentially growing Jurkat mHIV-Luciferase or mdHIV Clone 11 cells (3x107 cells/IP) were fixed with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature. Cross-linking was quenched with 125 mM glycine for 5 min, at which point cells were collected and washed with ice cold PBS. Cells were incubated in NP-40 Lysis Buffer (0.5% NP-40, 10 mM Tris-HCl pH = 7.8, 3 mM MgCl2, 1x PIC, 2.5 mM PMSF) for 15 min on ice. Following sedimentation, the supernatant was discarded, and the pellet resuspended in Sonication Buffer (10 mM Tris-HCl pH = 7.8, 10 mM EDTA, 0.5% SDS, 1x PIC, 2.5 mM PMSF). Nuclei were sonicated using a Covaris S220 Focused-ultrasonicator to produce sheared DNA between 2000-200 bp. Samples were pelleted, with the soluble supernatant collected as the chromatin fraction and snap frozen in liquid nitrogen. Chromatin concentrations were normalized among samples and pre-cleared with Protein A/G agarose (Millipore, 100 L/IP). The chromatin samples were split in two and diluted with IP buffer (10 mM Tris-HCl pH = 8.0, 1.0% triton X-100, 0.1% deoxycholate, 0.1% SDS, 90 mM NaCl, 2 mM EDTA, 1x PIC). The indicated specific antibody was added to one sample while control IgG was added to the other and the chromatin/ antibody mixture was incubated 1 hr at 40C with rotation. Antibodies used are as follows: TFII-I (10 μg) - BD Biosciences 610942, TRIM24 (10 μg) - Proteintech 14208-1-AP, RNAPII (5 μg) - Abcam ab26721, RNAPII pS2 (5 μg) - Abcam ab238146, CDK9 (10 μg) - Abcam ab239364, NFκB p65 (5 μg) - Thermo Fisher 51-0500, KAP1 (10 μg) - Abcam ab10483, H3K27ac (5 μg) – Abcam ab4729, H3K9me3 (5 μg) – Abcam ab176916, Mouse IgG (equivalent μg) – Santa Cruz sc-2025, Rabbit IgG (equivalent μg) – Abcam ab1722730. Pre-washed Protein A/G agarose beads (40 L/IP) were then added to the samples and incubated overnight at 40C with rotation. Bead – antibody complexes were washed 3x in Low Salt Wash Buffer (20 mM Tris-HCl pH = 8.0, 0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1x PIC) and 1x with High Salt Wash Buffer (same but with 500 mM NaCl). Elution and crosslink reversal was performed by incubating 4 hrs at 650C in EB supplemented with RNase A. DNA was purified using the QIAQuick PCR purification kit (QIAGEN) and ChIP DNA was analyzed using the Quant Studio 3 Real-Time PCR system (Applied Biosystems). Oligos used for ChIP-qPCR are as follows; RBE3/Nuc-0: Fwd 5’ AGCCGCCTAGCATTTCATC, Rev 5’ CAGCGGAAAGTCCCTTGTAG. RBE1/Nuc-1: Fwd 5’ AGTGGCGAGCCCTCAGAT, Rev 5’ 44 AGAGCTCCCAGGCTCAAATC. Gag: Fwd 5’ AGCAGCCATGCAAATGTTA, Rev 5’ AGAGAACCAAGGGGAAGTGA. NFκB Enhancer Region (NER): Fwd 5’ TTTCCGCTGGGGACTTTC, Rev 5’ CCAGTACAGGCAAAAAGCAG. 2.4.6 BioID assays 8.33x105 HEK293T cells were plated in 2 mL DMEM in 6-well plates and incubated overnight. Cells were transfected using PEI with 3 g of the indicated TurboID construct and incubated overnight. Biotin was added directly to the media at a final concentration of 500 M and cells were incubated for 1 hr. Biotinylation was stopped by washing cells 2x in ice cold PBS. Endogenous TRIM24 was immunoprecipitated as described above (2 μg of TRIM24 - Proteintech #14208-1-AP), and samples analyzed by immunoblotting. 2.4.7 Luciferase reporter assays Transient luciferase expression assays in HEK293T cells were performed as previously described (Dahabieh et al, 2011). Briefly, transfections were performed in 96 well plates seeded with 2x104 HEK293T cells per well 24 hours prior to transfection. 10 ng of pGL3 reporter plasmid along with 10 ng of either pcDNA3.1+ (Invitrogen) or pcDNA-Tat and 100 ng expression vector was co-transfected. Luciferase activity was measured 24 hours post transfection. For Jurkat luciferase reporter assays, 1x105 luciferase expressing Jurkat cells were plated with 100 µL media in 96-well plates. Luciferase activity was measured after the indicated time of treatment. Measurements were performed using SuperlightTM luciferase reporter Gene Assay Kit (BioAssay Systems) as per the manufacturer’s instructions; 96 well plates were read in a VictorTM X3 Multilabel Plate Reader. 2.4.8 Q-RT-PCR RNA was extracted from Jurkat mHIV-Luciferase cells following the indicated treatment using RNeasy Kit (Qiagen). RNA was analyzed using the Quant Studio 3 Real-Time PCR system (Applied Biosystems) using Power SYBR® Green RNA-to-CT™ 1-Step Kit (Thermo Fisher) as per the manufacturer’s instructions. RT-PCR data was normalized to GAPDH expression using the ΔΔCt method as previously described (Livak & Schmittgen, 2001). Cycling parameters were as follows: 48 oC, 30 min, 1x; 95 oC, 10 min, 1x; 95 oC, 15 sec, 60 oC, 1 min, 40x. Primers for analysis of HIV transcripts were as follows: Initiation, Fwd 5’ GTTAGACCAGATCTGAGCCT, Rev 5’ GTGGGTTCCCTAGTTAGCCA; Proximal, Fwd 5’ TGGGAGCTCTCTGGCTAACT, Rev 5’ TGCTAGAGATTTTCCACACTGA; Gag, Fwd 5’ CTAGAACGATTCGCAGTTAATCCT, Rev 5’ CTATCCTTTGATGCACACAATAGAG; GAPDH, Fwd 5’ TGCACCACCAACTGCTTAGC, Rev 5’ GGCATGGACTGTGGTCATGAG. 2.4.9 ChIP-seq analysis Call sets were downloaded from the ENCODE website (https://www.encodeproject.org/). Library identifiers are as follows; ENCLB915UWS, ENCLB381ZDN, ENCLB949IYC, 45 ENCLB887DPL. Heatmaps were generated from the ENCODE data using the plotHeatmap program. 2.4.10 RNA-seq RNA was extracted from wildtype, TRIM24 KO, LKO control shRNA transduced, or TFII-I shRNA transduced Jurkat T cells that were untreated or treated with 20 nM PMA/ 1 μM ionomycin for 4 hrs. Sample quality control was performed using a Agilent 2100 Bioanalyzer. Qualifying samples were then prepped following the standard protocol for the NEBnext Ultra ii Stranded mRNA (New England Biolabs). Sequencing was performed on the Illumina NextSeq 500 with Paired End 42bp × 42bp reads. De-multiplexed read sequences were uploaded to the Galaxy web platform (Afgan et al, 2018) and aligned to the hg38 reference genome using STAR; featureCounts determined transcript assembly, and differential gene expression (DEG) was determined by DESeq2 analysis. TRIM24 KO and TFII-I knockdown DEG are defined as having a fold change in expression > 1.5 and p-value < 0.05. The criteria for genes identified as responsive to T cell activation are fold change > 2, p-value < 0.05. Heatmaps were created in RStudio. 2.4.11 Cell adherence assay 2x106 wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were plated in 6-well plates with 2 mL RPMI. Cells were left untreated or treated with 20 nM PMA and 1 µM ionomycin and incubated for 2 hrs. The overlayed media was collected and the cells within were counted as the proportion of cells suspended. The cells that remained on the culture plates were scraped, resuspended in media, and counted as the adherent population, using a Bio-Rad TC20 Automated Cell Counter. 2.4.12 Flow cytometry Cells were treated as indicated in the figure legends. Following the indicated treatment, cells were suspended in PBS and analyzed on a BD Biosciences LSRII-561 system where threshold forward scatter (FSC) and side scatter (SSC) parameters were set so that a homogenous population of live cells were counted (Fig. S2.14). FlowJo software (TreeStar) was used to analyze data and determine mean fluorescent intensity (MFI). 2.4.13 Statistics and reproducibility All replicates are independent biological replicates and are presented as mean values with ± standard deviation shown by error bars. The number of times that an experiment was performed is indicated in the figure legends. P-values were determined by performing unpaired samples t-tests or two-way ANOVA, as indicated, with the use of GraphPad Prism 9.0.0. Statistical significance is indicated at *P < 0.05, **P < 0.01, or ***P < 0.001, with n.s. denoting non-significant P ≥ 0.05. 46 2.4.14 Data availability All data supporting the findings of this Chapter are available within the article Horvath et al, 2023 or from the corresponding author (I. Sadowski, ijs.ubc@gmail.com) upon reasonable request. All high-throughput RNA-seq data has been deposited to NCBI GEO and are publicly available under the accession codes GSE221633 and GSE221851. 2.5 Chapter acknowledgments We thank Andy Johnson and Justin Wong of the UBC Flow Cytometry Facility for performing FACS analysis as well as for assistance with flow cytometry. We thank the ENCODE Consortium and the Michael Snyder Lab for their work and for making the TFII-I and TRIM24 ChIP-seq data sets publicly available. ChIP-seq analysis of ENCODE data was performed by Stephane Flibotte of the UBC Life Sciences Institute Bioinformatics Facility, supported by the UBC GREx Biological Resilience Initiative. This research was supported by program project grant F16-01210, from the Canadian Institutes of Health Research. 2.6 Supplementary figures Figure S2.1 Effect of T cell activation on TFII-I and TRIM24 expression. A: Following 4 hr treatment of Jurkat mHIV-Luciferase cells with a vehicle control (Ve, DMSO) or 20 nM PMA/ 1 M ionomycin, whole cell lysates were analyzed by immunoblotting with antibodies targeting TFII-I, TRIM24, or Tubulin. B, C: Jurkat mHIV-Luciferase cells were incubated with a vehicle (Ve, DMSO) or 20 nM PMA/ 1 M ionomycin for 4 hrs. RNA was extracted and RNA-seq was performed (n = 3). Normalized counts of TFII-I (GTF2I, B) and TRIM24 mRNA (C) were produced by DESeq2 analysis. Statistical analysis was performed using unpaired t-test. 47 Figure S2.2 TurboID fusion proteins expressed in HEK293T cells. A: Schematic representation of Myc epitope tagged GFP and TFII-I TurboID fusions. B: HEK293T cells (lane 1) were transfected with plasmids expressing GFP-TurboID (lanes 3-6), TFII-I-TurboID (lanes 7-10), or an empty vector control (EV, lane 2). Cells were incubated with 500 µM biotin for the indicated amount of time (lanes 3-10), when lysates were prepared and immunoblotted using Streptavidin-HRP or anti-Myc antibodies. Figure S2.3 TRIM24 knockout inhibits HIV-1 expression. A: Knockout lines of TRIM24 (lanes 2-7) were produced from Jurkat mHIV-Luciferase cells (lane 1) using CRISPR/ Cas9. Following expansion of clones, lysates were analyzed by immunoblotting using antibodies against TRIM24 or Tubulin. B: Jurkat mHIV-Luciferase parent (WT) and TRIM24 KO clonal cell lines were treated with a vehicle control (Ve, DMSO) or with 20 nM PMA for 4 hr, at which point luciferase activity was measured (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 48 Figure S2.4 NFκB p65 is recruited to the LTR upon T cell activation. ChIP qPCR analysis with antibodies against NFκB p65 was performed with Jurkat Tat mdHIV Clone 11 following vehicle control treatment (Ve, DMSO) or stimulation with 50 nM PMA for 24 hrs. Results were normalized by subtraction of values produced with sample paired non-specific IgG (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. Figure S2.5 TRIM24 and TFII-I co-localize to the LTR in HeLa cells. A: TZM-bl cells were treated with DMSO or PMA (100 ng/mL) for 4 hr prior to measurement of luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. B: ChIP-qPCR with TZM-bl cells treated with DMSO (Ve) or PMA was performed using the indicated antibodies. n = 3, mean ± SD, with normalization performed by subtracting IgG values. Ve PMA0.000.050.100.15NFkB p65NFkB LTR SitesNormalized %Input✱✱49 Figure S2.6 TRIM24 depletion does not alter CDK9 or cyclin T protein levels. Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were left untreated (DMSO) (lanes 1-2) or treated with 20 nM PMA (lanes 3-4) for 4 hrs. Lysates were analyzed by immunoblotting with antibodies against CDK9, Cyclin T1, or Tubulin as indicated. Figure S2.7 CDK9 kinase activity is necessary for induction of HIV-1 expression. Following 1 hr pre-treatment with the indicated concentration of CDK9 kinase inhibitor LDC67, Jurkat mHIV-Luciferase cells were left untreated (DMSO) or incubated with 20 nM PMA for 4 hr prior to luciferase measurement (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. Ve PMA020406080100120%Un PMA Luciferase InductionUnVe0.5 μM1.0 μM2.5 μM5.0 μM10 μMnsns✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱✱50 Figure S2.8 TRIM24 regulates HIV-1 latency independently of TRIM28. A. ChIP-qPCR was performed on wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells using anti-TRIM28 antibody. Paired non-specific IgG values have been subtracted, n = 2, mean ± SD. Statistical analysis was performed using unpaired t-test. B: RNA-seq was performed on mRNA extracted from wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells. Shown are the TRIM28 mRNA normalized counts produced by DESeq2 analysis of 3 independent replicates (n = 3). Statistical analysis was performed using unpaired t-test. C: Wildtype or TRIM24 KO Jurkat mHIV-Luciferase cells were incubated with a vehicle control (Ve, DMSO) or 20 nM PMA/ 1 M ionomycin for 4 hrs. Subsequently, whole cell lysates were extracted and subject to immunoblotting with antibodies against TRIM28 or Tubulin. 51 Figure S2.9 Co-localization of TRIM24 and TFII-I in K562 cells. A: Venn diagram indicating overlap of ChIP-seq peaks derived from IP of eGFP-TFII-I or TRIM24 performed in K562 cells. B: Heatmap depiction of eGFP-TFII-I or TRIM24 binding sites obtained from ChIP-seq using the K562 cell line. c: Annotation of the genomic distribution of eGFP-TFII-I, TRIM24, and TFII-I/TRIM24 overlapping ChIP-seq peaks. 52 Figure S2.10 TRIM24 KO and TFII-I knockdown differentially expressed genes (DEG). A, B: Volcano plot depicting DESeq2 analysis comparing wildtype to TRIM24 KO (A) or control shRNA transduced cells to TFII-I shRNA transduced Jurkat mHIV-Luciferase cells (B). Samples were treated with 20 nM PMA/ 1 M ionomycin for 4 hrs prior to RNA extraction. Analysis was performed on n = 3 RNA-seq samples with significant genes having p-value < 0.05 and fold change > 1.5. 53 Figure S2.11 Loss of TRIM24 or TFII-I does not prevent global T cell activation. A, B, D, E: Volcano plot depiction of DEG resultant from 4 hr treatment with 20 nM PMA/ 1 M ionomycin of the indicated Jurkat mHIV-Luciferase cell lines. Genes that exhibited greater than 1.5-fold change and p-value < 0.05 upon PMA/ ionomycin treatment are defined as significant. DESeq2 analysis was performed on n = 3 RNA-seq samples. C, F: Venn diagram display of genes significantly repressed or activated by PMA and ionomycin treatment for the indicated cell line. 54 Figure S2.12 TRIM24 KO gene regulation is reflected by TFII-I knockdown. A, B: Heat map depictions of TRIM24 KO down-regulated (A) and up-regulated (B) genes. Relative gene expression was determined by DESeq2 analysis of n = 3, mean ± SD RNA-seq samples that were treated with 20 nM PMA/ 1 M ionomycin for 4 hrs prior to RNA extraction. 55 Figure S2.13 TRIM24 regulated cellular adhesion genes are similarly affected by TFII-I knockdown. A, B: Heat maps depicting the expression of TRIM24 KO down-regulated (A) and up-regulated (B) cellular adhesion genes. Relative gene expression was determined by DESeq2 analysis of n = 3 RNA-seq samples that were treated with 20 nM PMA/ 1 M ionomycin for 4 hrs prior to RNA extraction. 56 Figure S2.14 Representative flow cytometry gating strategy. Representative scatter plots depicting the gating strategy employed. Homogeneous populations of live cells were isolated and assessed by setting threshold forward scatter (FSC) and side scatter (SSC) settings. 57 58 59 Figure S2.15 Full length western blots. 60 Chapter 3: Inhibition of the TRIM24 bromodomain reactivates latent HIV-1 3.1 Introduction Reactivation of HIV-1 expression in response to TCR signaling requires multiple factors specifically bound to the 5' LTR, including TFII-I which is constitutively associated with two highly conserved cis-elements flanking the LTR enhancer region in association with USF1/2 (Chen et al, 2005) (Dahabieh et al, 2011). As described in Chapter 2, we discovered that TFII-I recruits the coactivator protein Tripartite-Motif containing protein 24 (TRIM24) to the LTR, and this interaction promotes transcriptional elongation from the LTR core promoter by RNA Polymerase II, an effect that is associated with enhanced recruitment of CDK9 and increased RNA Pol II CTD on serine 2 phosphorylation (Horvath et al, 2023). These observations indicate that TRIM24 may function as a transcriptional coactivator by promoting recruitment of P-TEFb. In this study we examine the effect of the TRIM24 bromodomain inhibitor IACS-9571 on HIV-1 transcription, and surprisingly, find this compound causes reactivation of latent provirus in multiple different T cell line models of provirus latency, and produces synergistic effects in combination with T cell signaling agonists. Treatment of T cells with IACS-9571 inhibits production of immediate latent provirus and encourages productive expression of HIV-1 in newly infected cells. IACS-9571 treatment caused elevated association of TRIM24 with the LTR suggesting that inhibition of the bromodomain may cause redistribution of this factor from cellular genes. Treatment of CD4+ T cells from individuals with HIV-1 on ART with IACS-9571 in combination with Ingenol 3-angelate (PEP005), a small molecule protein kinase C (PKC) activator (Kedei et al, 2004), resulted in robust viral transcription while also suppressing induction of several T cell activation markers including IL2 and CD69. These results indicate that the TRIM24 bromodomain inhibitor IACS-9571 represents a novel class of latency reversing agent which may prove useful for strategies to purge latently infected cells. 3.2 Results 3.2.1 The TRIM24 bromodomain inhibitor IACS-9571 promotes HIV-1 expression We discovered that TRIM24 is recruited to the HIV-1 LTR by interaction with TFII-I, and this causes enhanced transcriptional elongation from the HIV-1 LTR (Horvath et al, 2023). Consequently, we examined what effect an inhibitor of the TRIM24 bromodomain might have on expression of HIV-1, using a Jurkat Tat mHIV-Luciferase cell line which bears an HIV-1 mini-virus where luciferase is expressed as a fusion with Gag (Fig. 3.1A) (Bernhard et al, 2011) (Horvath et al, 2023). Surprisingly, IACS-9571 caused activation of luciferase expression in a dose-dependent manner, where treatment with 25 M caused significant 3-fold induction in otherwise untreated cells (Fig. 3.1B), at concentrations that produce minimal toxicity (Fig. 3.1C). A similar effect of IACS-9571 was observed using JLat10.6 cells, which bear an HIV-1 provirus where Nef has been replaced by GFP (Fig. S3.1). 61 We also examined the effect of IACS-9571 on newly infected using a dual reporter Red-Green HIV (RGH) which enables detection of infected cells by expression of mCherry from an internal PGK promoter, and independent measurement of expression from the 5' LTR with a GFP reporter (Fig. 3.1D) (Dahabieh et al, 2013). Productively infected cells express both mCherry and GFP, whereas infected cells that produce immediate latent provirus only express mCherry (Fig. 3.1D). RGH infected cells treated with PMA and ionomycin 4 days post-infection produced ~70% productive infections, a significant increase from the ~40% productive infections in untreated (Ve) treated cells (Fig. 3.1E). IACS-9571 also caused an increase in the proportion of productively infected cells compared to the untreated control (Fig. 3.1E), indicating activation of latent provirus in a significant proportion of newly infected T cells. Next, we examined the effect of IACS-9571 on virus expression over 10 days post infection where we found that ~35% of infected untreated cells (Ve) developed productive infections at 4 days post infection; the proportion of productively infected cells decayed from 4-10 days as more cells established latency by epigenetic mechanisms (Fig. 1F) (Hashemi et al, 2016). In contrast, cells treated with IACS-9571 generated a significantly greater proportion of productively infected cells, such that ~50% of infected cells expressed GFP 4 days post-infection (Fig. 3.1F, IACS-9571). Throughout the course of treatment, negligible effects upon cell viability were observed (Fig. 3.1G). These observations indicate that the TRIM24 bromodomain inhibitor dissuades establishment of immediate latency in newly infected cells and forces productive expression of viral RNAs from the 5' LTR. 62 Figure 3.1 IACS-9571 promotes HIV-1 expression. A: Schematic representation of HIV-1 mini-virus in the Jurkat Tat mHIV-Luciferase cell line, where luciferase is expressed from the 5' LTR as a fusion with Gag. B: mHIV-Luciferase cells were treated with the indicated concentration of IACS-9571 for 4 hrs prior to luciferase assay. Results are an average of three determinations, and error bars represent standard deviation. C: mHIV-Luciferase cell viability was determined after 4 hrs treatment with IACS-9571 at the indicated concentration. Viability assays were performed in duplicate with error bars representing standard deviation shown. D: Depiction of Red-Green-HIV-1 (RGH) reactivation assays. RGH is a full-length replication incompetent HIV-1 virus with GFP expressed by the 5’ LTR as a fusion with Gag and Nef is replaced with a PGK promoter that drives mCherry expression (Dahabieh et al, 2013). Wildtype 63 Jurkat T cells were infected with RGH at a low MOI; 4 days post-infection (p.i.), pools of cells were treated with 10 µM IACS-9571, 20 nM PMA/ 1 µM ionomycin, or left untreated for 24 hrs. Subsequently, productive and latently infected cells were analyzed by flow cytometry. E: Ratio of productively infected cells following RGH reactivation. Results are an average of two determinations, and error bars represent standard deviation. Statistical analysis was performed using unpaired t-test. F: Wildtype Jurkat T cells infected with RGH were treated with or without 10 µM IACS-9571 throughout the course of infection. Flow cytometry was performed on the indicated day post-infection (p.i.). Shown are the results of duplicate experiments, and error bars represent standard deviation. Statistical analysis was performed using unpaired t-test. G: Cell viability was determined at each time post-infection. Results of two measurements are shown, with error bars representing standard deviation. 3.2.2 IACS-9571 stimulates HIV-1 expression in primary CD4+ T cells ex vivo We also examined the effect of IACS-9571 on HIV-1 replication in primary CD4+ T cells isolated from peripheral blood mononuclear cells (PBMCs). Here, we infected CD4+ T cells with the RGH dual report virus while treating with IACS-9571 or a vehicle control (Fig. 3.2A). Consistent with results using T cell lines, IACS-9571 caused an increase in the proportion of productive infections in normal CD4+ T cells (Fig. 3.2B, 3.2C). Interestingly, productive infections established during IACS-9571 treatment produced stronger expression of GFP from the 5' HIV-1 LTR than provirus established in the absence of the drug (Fig. 3.2C, Productive). Notably, no change in CD4+ T cell viability was observed as a result of IACS-9571 treatment (Fig. 3.2D). These results confirm that IACS-9571 stimulates HIV-1 transcription in primary T cells. 64 Figure 3.2 IACS-9571 regulates HIV-1 latency in primary CD4+ T cells. A: Schematic representation of the experimental design. CD4+ T cells were pre-treated for one hour with a vehicle control or 10 μM IACS-9571 and subsequently infected with RGH. Following three days of continued treatment, flow cytometry was performed. B: Determination of the ratio of productively infected CD4+ T cells following 3 days of treatment with 10 μM IACS-9571 or vehicle control. Results of three biological measurements are shown, with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. C: As in B but evaluating the Mean Fluorescence Intensity (MFI) of GFP expression. The GFP MFI was evaluated within the entire population of infected cells (All Infections) or within the population of cells that harbored productive infections (Productive). Statistical analysis was performed using unpaired t-test. D: Cells were treated as in A and B, and viability was measured. Measurements were performed in duplicate with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. 3.2.3 IACS-9571 produces synergistic effects with various LRAs HIV-1 transcription is activated by multiple signaling pathways regulated by engagement of the T cell receptor and cytokines, including TNF- and IL2 (Mbonye & Karn, 2014) (Sadowski & Mitchell, 2005), which control transcriptional activators bound to the LTR, including NF-B, AP1, GABP/ Ets, NFAT, and USF1/2-TFII-I (RBF-2) (Malcolm et al, 2007) 65 (Sadowski & Mitchell, 2005). We examined how IACS-9571 might influence responses to these signaling mechanisms by treatment in combination with signaling agonists. Jurkat mHIV-Luciferase cells (Fig. 3.1A) were treated with 10 µM IACS-9571 and luciferase expression was measured over the next 24 hours, where we observed a modest, but significant ~2-fold induction of luciferase expression at 6 hours (Fig. 3.3A). However, this concentration of IACS-9571 produced elevated HIV-1 expression in combination with any of the signaling agonists we examined. This includes PMA (Fig. 3.3B, 3.3D,), which causes activation of multiple factors (NF-B, AP1, GABP/ Ets); Ionomycin (Fig. 3.3C, 3.3D), which activates NFAT; TNF- (Fig. 3.3E), which stimulates NF-B and AP1; PEP005 (Fig. 3.3F), an LRA which acts through NF-B; and JQ1, a BRD4 bromodomain inhibitor (Fig. 3.3G). Additionally, co-treatment with a combination of IACS-9571 and the HDAC inhibitor SAHA produced a modest increase in LTR-luciferase expression up to 6 hrs, an effect that is not only lost but ostensibly antagonized after 24 hrs (Fig. 3.3H). We examined the combined effect of IACS-9571 and the LRAs on stimulation of expression using Bliss independence modeling (Laird et al, 2015) (Liu et al, 2018), which indicated that IACS-9571 produced a synergistic effect with all of the T cell signaling agonists examined (Fig. S3.2). Notably, IACS-9571 and PEP005 induced the largest response of HIV-1 expression (Fig. 3.3F), and also produced the greatest degree of synergy amongst the LRAs examined (Fig. S3.2H). Co-treatment of IACS-9571 and SAHA produced a slight synergistic effect at 4 and 6 hours with an antagonistic interaction emerging following 24 hours (Fig. S3.2G). These observations are consistent with previous experiments demonstrating synergy between various LRAs that function through independent mechanisms (Hashemi & Sadowski, 2020). 66 Figure 3.3 IACS-9571 produces activation in combination with T cell signaling agonists. A - H: Jurkat Tat mHIV-Luciferase cells were treated with the indicated latency reversing agent, with or without 10 µM IACS-9571. Concentration of agonist used were 20 nM PMA, 1 µM ionomycin, 10 ng/µL TNFα, 20 nM PEP005, 10 μM JQ1, 10 μM SAHA. Luciferase assays were 67 performed at the indicated times. Results of three determinations are displayed, with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. 3.2.4 TRIM24 is necessary for activation of HIV-1 expression, including by IACS-9571 The TRIM24 bromodomain has similar structure to that of another protein BRPF1, and IACS-9571 was shown to bind these bromodomains with similar affinity (Palmer et al, 2016). Consequently, it is possible that the effect of IACS-9571 on the HIV-1 LTR may be mediated by factor(s) other than TRIM24, including potentially BRPF1 (Palmer et al, 2016). To examine this, we compared the effect of IACS-9571 on HIV-1 provirus expression in the mHIV-Luciferase Jurkat cell line (Fig. 3.1A), and a derivative line bearing a CRISPR/ Cas9-mediated gene disruption of TRIM24 (Fig. 3.4, TRIM24 KO) (Horvath et al, 2023). We observe dose-dependent induction in WT cells treated with IACS-9571, but this effect is completely inhibited in cells bearing the TRIM24 KO (Fig. 3.4A). A similar effect was observed in cells treated with both IACS-9571 and PMA (Fig. 3.4B), where we observed a synergistic effect on HIV-1 expression in WT cells, but reactivation is significantly inhibited in the TRIM24 KO line, with only slight activation at the highest concentration of IACS-9571. These results indicate that TRIM24 is the primary mechanistic target of IACS-9571 for induction of HIV-1 expression. To further delineate effects of TRIM24 and BRPF1 bromodomain inhibition on HIV-1 expression, we treated mHIV-Luciferase Jurkat cells with GSK-5959, a potent and selective BRPF1 bromodomain inhibitor (Demont et al, 2014). Unlike treatment with IACS-9571, we did not observe a dose-dependent effect on HIV-1 expression, however 10 μM GSK-5959 produced a modest but statistically significant 1.5-fold increase in HIV-1 expression in otherwise untreated cells (Fig. 3.4C, Ve). We also examined the effect of GSK-5959 in combination with PMA but found that unlike IACS-9571, this co-treatment failed to elevate HIV-1 expression relative to PMA alone (Fig. 3.4C, PMA). Of note, the concentrations of GSK-5959 applied did not affect cellular viability (Fig. 3.4D). These results are consistent with those presented above with TRIM24 knockout cells, and we conclude that inhibition of the BRPF1 bromodomain by IACS-9571 does not contribute to reactivation of latent HIV-1. The small effect of IACS-9571 in combination with PMA in the TRIM24 knockout cell line (Fig. 3.4B) may reflect residual off-target effects. All bromodomain inhibitors developed to date, including JQ1 (BRD4), GSK-5959 (BRPF1), and IACS-9571 (TRIM24/BRPF1) produce some off-target effects. Interestingly, IACS-9571 and GSK-5959 share similar off-targets including BRPF2/ BRD1, BRPF3, BAZ2B, and TAF1(Demont et al, 2014) (Palmer et al, 2016), although GSK-5959 does not bind the TRIM24 bromodomain (Demont et al, 2014). Because GSK-5959 did not affect HIV-1 latency (Fig. 3.4C), it seems unlikely that the major effect of IACS-9571 on HIV-1 expression is produced by off target effects on additional bromodomain proteins. 68 Figure 3.4 IACS-9571 targets TRIM24 to induce expression of HIV-1. A: Wildtype and TRIM24 KO mHIV-Luciferase cells were treated with the indicated concentration of IACS-9571 for 4 hrs prior to luciferase assay. Assays were performed in triplicate, with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. B: Same as A, but cells were co-treated with 20 nM PMA. Shown are the results of triplicate measurements, error bars represent standard deviation. Statistical analysis was performed using unpaired t-test. C: Jurkat mHIV-Luciferase cells were treated with the indicated concentration of GSK 5959 in the presence of absence of 20 nM PMA. Following 4 hrs of incubation, luciferase assay was performed. Shown are the results of three measurements, with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. D: Viability of mHIV-Luciferase cells were measured following 4 hrs incubation with the indicated concentration of GSK 5959. Assay was performed in duplicate with standard deviations displayed as error bars. 3.2.5 PROTAC-induced degradation of TRIM24 inhibits HIV-1 expression To examine the requirement of TRIM24 for induction of HIV-1 we used a Von Hippel-Lindau (VHL)-engaging functional degrader of TRIM24, designated dTRIM24 (Gechijian et al, 2018). This chemical derivative is comprised of IACS-9571 conjugated to the VHL binding 69 ligand VL-269 to produce a proteolysis-targeting chimeric compound (PROTAC), which promote degradation of target proteins by forcing interaction with the VHL ubiquitin ligase (Gechijian et al, 2018). Treatment of Jurkat T cells bearing a mini-dual HIV-1 reporter provirus (Hashemi et al, 2016) (Fig. 3.5A) with dTRIM24 causes degradation of TRIM24 at concentrations between 0.5 - 5 mM after 24 hours, as determined by immunoblotting (Fig. 3.5B, lanes 2-5). As with previous observations, dTRIM24 mediated degradation is lost at higher concentrations (Fig. 3.5B, lane 6), an effect that is proposed to result from the molecule favoring binary interaction over ternary complex formation (Gechijian et al, 2018). Having verified dTRIM24 concentrations that produce its degradation, we measured induction of HIV-1 provirus treated cells by analyzing dsRed expression (Fig. 3.5A) 20 hours following stimulation with PMA. Consistent with our previous results indicating that TRIM24 is required for reactivation of latent HIV-1 (Horvath et al, 2023), we found that cells treated with concentrations of dTRIM24 that reduce TRIM24 protein levels also displayed a corresponding decrease in expression of the 5' LTR dsRed reporter in these cells (Fig. 3.5C), but without significantly affecting cell viability at effective concentrations (Fig. 3.5D). Notably, a linear correlation between TRIM24 protein level and LTR induction did not occur. This is likely a consequence of opposing effects caused by dTRIM24; dTRIM24 mediated degradation of TRIM24 inhibits expression from the LTR, whereas dTRIM24 inhibition of the TRIM24 bromodomain causes activation. These results demonstrate that the TRIM24 bromodomain inhibitor IACS-9571 produces a differential effect on HIV-1 transcription than does complete inhibition of TRIM24 protein function by factor depletion. 70 Figure 3.5 Effect of dTRIM24 on HIV-1 expression. A: Schematic representation of HIV-1 mini-virus integrated in the Clone #11 Jurkat Tat cell line, where dsRed is expressed from the 5' LTR, while GFP is driven by the constitutive EIF1α promoter (Hashemi et al, 2016). B: Clone #11 cells were treated with the indicated concentration of dTRIM24 or left untreated (Ve) for 24 hrs. Immunoblots were performed on whole cell lysates, with antibodies against TRIM24 (top) or Tubulin (bottom). C: Following 4 hr pre-treatment with the indicated concentration of dTRIM24, Clone #11 cells were stimulated with 40 nM PMA for 20 hrs and analyzed by flow cytometry. Assays were performed in duplicate, with error bars representing standard deviation displayed. Statistical analysis was performed using unpaired t-test. D: Cell viability was measured in cells as treated in C. Results are an average of two determinations, and error bars represent standard deviation. 3.2.6 The C-terminal TRIM24 bromodomain is dispensable for activation of HIV-1 expression Because the IACS-9571 TRIM24 bromodomain inhibitor produces the opposite effect as TRIM24 depletion, we examined whether the C-terminal bromodomain of TRIM24 was necessary for activation of HIV-1 expression. For this, we co-transfected HEK293T cells with vectors expressing WT TRIM24, or TRIM24 mutants bearing ORF deletions and point mutations in the C-terminal PHD-bromodomain motifs (Fig. 3.6A, B) in combination with an HIV-1 LTR-luciferase reporter. Using this assay, we have previously demonstrated that the effect of TRIM24 on HIV-1 expression does not require Tat (Horvath et al, 2023). Co-transfection of WT TRIM24 expression plasmid causes ~3-fold stimulation of HIV-1 luciferase expression (Fig. 3.6B, TRIM24). Interestingly, this effect of TRIM24 does not require the C-terminal PHD-bromodomain region, as a deletion lacking the entire C-terminus (Fig. 3.6B, C-Terminus) causes the same effect as WT TRIM24. Similarly, derivatives bearing amino acid substitutions in the C-terminal PHD-bromodomain motifs (F979A, N980A, C840W) that disrupt chromatin binding cause comparable levels of HIV-l expression as WT (Fig. 3.6B). In contrast, deletion of the complete N-terminus or the coiled-coil motif (Fig. 3.6C) prevented stimulation of HIV-1 expression (Fig. 3.6D). A mutant with deletion of the BB2 motif was less effective for stimulation of HIV-1 expression (Fig. 3.6D), but we note that this protein is expressed at significantly lower levels than the other TRIM24 derivatives (Fig. 3.6C). These results indicate that the bromodomain motif is not required for the effect of TRIM24 for reactivation of HIV-1 transcription. 71 Figure 3.6 TRIM24 chromatin binding is dispensable for activation of HIV-1 transcription. A, C: Two days post-transfection of HEK293T cells with the indicated construct, whole cell lysates were collected and immunoblotted using antibodies against Myc (top) or tubulin (bottom). B, D: HEK293T cells were co-transfected with LTR-Luciferase reporter construct and the indicated expression vector. Two days post-transfection, LTR expression was analyzed by luciferase assay. Results are the average of three determinations with standard deviation indicated. Statistical analysis was performed using unpaired t-test. 3.2.7 TRIM24 LTR occupancy is enhanced by IACS-9571 We have previously shown that TRIM24 is associated with the HIV-1 LTR by direct interaction with TFII-I bound to the RBE3 and RBE1 cis-elements, positioned at -120 relative to the transcriptional start site, and immediately adjacent the core promoter, respectively (Horvath et al, 2023). The TRIM24 C-terminal tandem bromodomain – plant homeodomain (PHD) is 72 capable of binding histones with preference for H3K23ac and H3K4me0 marks, respectively (Groner et al, 2016) (Tsai et al, 2010). Consequently, because TRIM24 is presumed to be predominantly recruited to chromatin via interactions mediated by the C-terminal bromo/ PHD domains, we examined the effect of bromodomain inhibition on recruitment of TRIM24 to the HIV-1 LTR. For this, we expressed Flag tagged TRIM24 in Jurkat cells harboring an HIV-1 reporter virus (Fig. 3.7A, lanes 2 and 3). Using ChIP-qPCR, we observed significantly enhanced interaction of TRIM24 with the LTR, at both RBE3 and RBE1 in cells treated with IACS-9571 (Fig. 3.7B). This observation is consistent with previous results indicating that TRIM24 is a limiting co-factor for activation of HIV-1 expression (Horvath et al, 2023), and suggests that inhibition of the TRIM24 bromodomain may inhibit global interaction with chromatin, thereby increasing the pool of TRIM24 available for recruitment by TFII-I at the HIV-1 LTR. Figure 3.7 Association of TRIM24 with the HIV-1 LTR is enhanced upon IACS-9571 treatment. A: Jurkat mHIV-Luciferase cells were transduced with lentivirus expressing Flag tagged TRIM24 (T24-Flag, Lanes 2, 3) or an empty vector (EV, Lane 1). TRIM24-Flag transduced cells were treated with 10 µM IACS-9571 (Lane 3) or left untreated (Ve, Lane 2). Whole cell lysates were immunoblotted using the indicated antibody. B: TRIM24-Flag transduced cells were left untreated or incubated with 10 µM IACS-9571. Following 4 hrs, ChIP was performed using the indicated antibody. Results are the average of qPCR performed in replicate with standard deviation shown. Statistical analysis was performed using unpaired t-test. 3.2.8 IACS-9571 stimulates HIV-1 transcriptional elongation Our previous results indicate that TRIM24 causes enhanced elongation of transcription by RNA Polymerase II from the viral promoter (Horvath et al, 2023). Consequently, we examined whether treatment of cells with IACS-9571 also stimulates transcriptional elongation. We found that treatment of Jurkat mHIV-Luciferase cells (Fig. 3.1A) with IACS-9571 did not cause enhanced recruitment of RNAPII to the LTR, rather we observe a slight decrease in RNA PolII occupancy at the LTR in otherwise untreated cells (Fig. 3.8A, compare Ve vs IACS-9571). Initiation of eukaryotic transcription is associated with phosphorylation of RNAPII at the C-73 terminal domain (CTD) S5, a modification mediated by CDK7 of TFIIH. As with total RNA Pol II occupancy, we observed slightly less pS5-modified RNAPII following IACS-9571 treatment as compared to untreated cells (Fig. 3.8B). Elongation of transcription from the LTR is stimulated by recruitment of P-TEFb, containing CDK9 which phosphorylates RNAPII CTD S2, among other factors, to promote transition from a paused to elongating transcription complex. Interestingly, in contrast to RNAPII and CTD pS5, we found that treatment of mHIV-Luciferase cells with IACS-9571 causes elevated association of RNAPII pS2 with the LTR as compared to untreated cells (Fig. 3.8C). Additionally, IACS-9571 treatment caused significant enrichment of CDK9 (P-TEFb) at the HIV-1 core promoter (Fig. 3.8D, RBE1). These results are consistent with previous observations indicating that recruitment of TRIM24 to the HIV-1 LTR causes enhanced recruitment of P-TEFb/ CDK9, which promotes RNAPII CTD S2 phosphorylation and elongation of transcription (Horvath et al, 2023). Results shown above (Fig. 3.3) indicate that IACS-9571 produces synergistic effects of HIV-1 provirus expression in combination with T cell signaling agonists. We note that treatment with PEP005, an agonist of the PKC pathway that is stimulated by the T cell receptor (Sadowski & Mitchell, 2005), causes significantly enhanced recruitment of RNAPII (Fig. 3.8A) and phosphorylation of CTD S5 (Fig. 3.8B) to the HIV-1 LTR, indicating that activation of factors regulated by T cell signaling have a significant effect on recruitment of RNA Pol II to the LTR promoter for promotion of transcriptional initiation. Co-treatment of cells with IACS-9571 and PEP005 causes corresponding elevated association of RNAPII, CTD pS5, CTD pS2, and CDK9 with the LTR (Fig. 3.8) which is consistent with the synergistic effect on HIV-1 reporter expression produced by the combination of these treatments. 74 Figure 3.8 IACS-9571 stimulates elongation from the HIV-1 LTR. A-D: Jurkat mHIV-Luciferase cells were treated with 10 µM IACS-9571, 20 nM PEP005, 10 µM IACS-9571 and 20 nM PEP005, or the equivalent amount of DMSO (Ve). Following 4 hrs treatment, ChIP was performed using the indicated antibody. Displayed are the average of replicate qPCR determinations with error bars representing standard deviation. Statistical analysis was performed using two-way ANOVA. 3.2.9 Effect of IACS-9571 on HIV-1 provirus expression in CD4+ T cells from individuals on ART To determine whether IACS-9571 was capable of affecting expression of HIV-1 provirus in primary CD4+ lymphocytes, we examined the effect of treatment on CD4+ T-cells isolated from individuals with HIV-1 on ART. We found that treatment with 10 μM IACS-9571 on its own did not produce a detectable increase in HIV-1 mRNA in cell samples from 6 participants examined (Fig. 3.9, compare Ve and IACS-9571). This observation is not surprising, considering that CD4+ T cells containing integrated, replication competent provirus, are outnumbered by uninfected cells from individuals on ART by several orders of magnitude (Sadowski & Hashemi, 2019). We examined the effect of the PKC agonist PEP005 (Hashemi & Sadowski, 2020) on HIV-1 expression in CD4+ T cells from these same donors, where we observed a significant 75 increase in viral transcription in cells from two of the participants (Fig. 3.9A, 3.9E, BC003 and BC008), and a more modest effect on a third participant (Fig. 3.9D, BC006). Interestingly, with cells from all of the samples, co-treatment with IACS-9571 and PEP005 caused significant induction of HIV-1 mRNA (Fig. 3.9), a result that is consistent with the synergistic effects these agents have on HIV-1 reporter expression in T cell lines (Fig. 3.3F, Fig. S3.2E). Figure 3.9 IACS-9571 and PEP005 synergistically activate HIV-1 expression in PBMCs. A - F: CD4+ PBMCs isolated from HIV-1 patients on ART were treated with DMSO (Ve), 10 µM IACS-9571, 15 nM PEP005, or with 10 µM IACS-9571 and 15 nM PEP005 for 20 hrs. Following incubation, intracellular RNA was extracted and subject to RT-PCR using oligos targeting HIV-1 mRNA. The results of two to three RT-PCR determinations are displayed with standard deviation depicted as error bars. Statistical analysis was performed using one-way ANOVA. 76 3.2.10 IACS-9571 partially suppresses T cell activation An important consideration for development of LRAs as potential therapies is that treatment should produce robust and broad induction of HIV-1 expression without causing global T cell activation. To determine the potential effect of IACS-9571 on T cell activation, we examined expression of IL2 and CD69 mRNA in cells from participants using RT-PCR. Consistent with previous results (Jiang et al, 2015), treatment of CD4+ cells from participants with HIV-1 on ART with the PKC agonist PEP005 caused a significant increase in IL2 and CD69 mRNA (Fig. 3.10A, 3.10B, compare Ve vs PEP005). In contrast, treatment with IACS-9571 on its own did not cause elevation of either IL2 or CD69 transcript abundance. Moreover, we observed that treatment with IACS-9571 in combination with PEP005 resulted in robust suppression of IL2 induction while also generating a more modest ~40% reduction in CD69 mRNA (Fig. 3.10A, 3.10B, compare PEP005 vs PEP005/IACS-9571). Notably, the effect of IACS-9571 on HIV-1 latency reversal and T cell activation are independent of toxicity, as the treatments did not impact viability (Fig. 3.10C). To further investigate the effect of IACS-9571 on T cell activation, we used the Jurkat T cell line to examine mRNA expression of T cell activation-induced genes. Similar to results with participant CD4+ samples, we found that IACS-9571 restricted IL2 and CD69 mRNA induction in response to T cell stimulation by PMA (Fig. 3.10D). Similarly, IACS-9571 inhibited PMA-induced expression of the cytokines CXCL10 and IL8 (Fig. 3.10D), but did not inhibit induction of IFN-γ, CSF2, or CCL22 transcripts, indicating that this bromodomain inhibitor causes selective partial inhibition of T cell activation induced genes. We also examined the effect of IACS-9571 on expression of the CD69 T cell activation surface marker by flow cytometry (Fig. S3.4). Consistent with analysis of mRNA, PMA treatment caused accumulation of surface associated CD69 (Fig. 3.10E, 3.10F), but this effect was inhibited in cells treated with PMA in combination with IACS-9571 (Fig. 3.10E). Although surface associated CD69 was drastically reduced by IACS-9572 (Fig. 3.10E), the majority of cells still presented with CD69 (Fig. 3.10F), indicating that IACS-9571 causes a uniform decrease in CD69 expression rather than completely suppressing a limited segment of the cell population (Fig. S3.4C). Collectively, these results indicate that IACS-9571 causes reactivation of HIV-1 expression while partially suppressing T cell activation. This divergent functionality represents an important feature as no previous LRA was found to produce opposite effects for T cell activation and induction of HIV-1 expression (Sadowski & Hashemi, 2019). 77 Figure 3.10 IACS-9571 does not cause T cell activation. A, B: CD4+ PBMCs isolated from ART administered HIV-1 patients were treated with DMSO (Ve), 10 µM IACS-9571, 15 nM PEP005, or with 10 µM IACS-9571 and 15 nM PEP005 for 20 hrs. Following treatment, intracellular RNA was extracted and subject to RT-PCR using oligos that target IL2 (A) or CD69 (B) mRNA. For each patient sample, RT-PCR was performed in replicate. Statistical analysis was performed using two-way ANOVA. C: Viability of participant CD4+ PBMCs was assessed following treatment as in A and B. A Bio-Rad TC20 Automated Cell Counter was used to measure viability. Each participant sample displayed is the average of two independent measurements. Statistical analysis was performed using two-way ANOVA. D: Jurkat mHIV-Luciferase cells were incubated with DMSO (Ve), 10 μM IACS-9571, 20 nM PMA, or a combination of 10 μM IACS-9571 and 20 nM PMA for 4 hrs. Following treatment, intracellular mRNA was isolated and RT-PCR was performed using oligos specific to the indicated transcript. Shown are the results of triplicate RT-PCR measurements with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. E, F: Jurkat mHIV-Luciferase cells were treated as in Panel D. Following treatment, surface associated CD69 was stained using a PE-Cy7 conjugated antibody and flow cytometry was performed, measuring either the Mean Fluorescent Intensity of CD69 - PE-Cy7 (E) or the percent of cells positive for surface CD69 78 presentation (F). Assays were performed in duplicate with standard deviations represented by error bars. Statistical analysis was performed using one-way ANOVA. 3.3 Discussion TRIM24 was initially identified as co-activator for various nuclear receptors (Thénot et al, 1997). This factor has E3 ubiquitin ligase activity, conferred by the N-terminal RING motif, and interacts with chromatin via the C-terminal PHD-bromodomain through binding of histone H3K4me0/H3K23ac (Tsai et al, 2010), but a mechanism for its function in transcriptional regulation had not been identified. We discovered that TRIM24 is recruited to the HIV-1 LTR by interaction with TFII-I, which promotes elongation of transcription in stimulated T cells through enhanced recruitment of P-TEFb/ CDK9 and phosphorylation of RNAPII CTD S2 (Horvath et al, 2023). Consequently, we were surprised that the TRIM24 bromodomain inhibitor IACS-9571 caused elevated expression of HIV-1 provirus in otherwise untreated Jurkat T cells, an effect accompanied by enhanced association of TRIM24 with the promoter. Because TRIM24 seems to be a limiting factor for reactivation of HIV-1 transcription (Horvath et al, 2023), our observations suggest that IACS-9571 may inhibit global interaction of this factor with chromatin and consequently increases opportunity for recruitment to the LTR by TFII-I. Global effects on the transcriptome by IACS-9571 are largely divergent from that caused by dTRIM24 facilitated protein degradation (Gechijian et al, 2018). In this study, we discovered HIV-1 expression is differentially affected by IACS-9571 and dTRIM24 treatment. IACS-9571 causes reactivation of HIV-1 provirus at similar concentrations, and as effectively, as most previously characterized LRAs (Hashemi & Sadowski, 2019). Conversely, we found that forced degradation of TRIM24 with dTRIM24 (Gechijian et al, 2018) inhibited HIV-1 expression at concentrations where it caused reduction of TRIM24 protein levels. This latter observation, in combination with our previous results indicating that TRIM24 KO inhibits HIV-1 reactivation (Horvath et al, 2023) suggests that full inhibition of TRIM24 function, or at least interaction with TFII-I on the LTR, represent prospective latency promoting targets. Overall, these observations indicate that TRIM24 is an important potential target than can be modulated to enhance or repress HIV-1 provirus expression for therapeutic strategies. The latency reversing effect of IACS-9571 may be produced by inhibition of its interaction with H3K4me0/H3K23ac modified chromatin on cellular genes. This effect might provide higher levels of TRIM24 available for recruitment to the LTR. Consistent with this view we observe enhanced levels of TRIM24 associated with the LTR in cells treated with IACS-9571 (Fig. 3.7). Furthermore, we find that TRIM24 mutant proteins bearing deletion of the C-terminal region spanning the histone binding motifs, or point mutations within those motifs, do not affect the capability of this factor to stimulate HIV-1 transcription (Fig. 3.6A, 3.6B). These observations incidentally suggest that the histone binding function of TRIM24 is not directly required for its effect on stimulating transcriptional elongation from the HIV-1 LTR. However, a more detailed 79 understanding of TRIM24 function will be required to elucidate the mechanism for IACS-9571 as an LRA. CD4+ T-helper 2 cells lacking TRIM24 display dampened inflammation characterized by reduced expression of many cytokines and chemokines (Perez-Lloret et al, 2016). Here, we found that unlike most other LRAs, IACS-9571 similarly inhibits expression of genes associated with T cell activation in CD4+ cells from individuals with HIV-1 on ART, and in Jurkat T cells stimulated with PMA. An important consideration for LRA development is their effectiveness for reactivation of HIV-1 provirus without causing global T cell activation (Sadowski & Hashemi, 2019). Unchecked activation of T cells can produce a response known as cytokine storm, typified by excess production of proinflammatory cytokines which can cause acute respiratory distress syndrome. Consequently, cytokine storm caused by hyperactivation of T cells contributes significantly to pathology of COVID-19 (Jiang et al, 2022) and influenza (Gu et al, 2021). Our observation that IACS-9571 inhibits genes associated with T cell activation but causes activation of HIV-1 illustrates a unique capability of this novel LRA. The differential effect of TRIM24 for regulation of HIV-1 and T cell response must be mediated by differential requirement for function of the C-terminal bromodomain. A more detailed understanding of the mechanistic significance of histone H3K23 acetylation will be required to elucidate the effect of IACS-9571 on HIV-1 transcription. Nevertheless, considering the unique effects this compound has on HIV-1 provirus and T cell activation we suggest that IACS-9571, and its derivatives, may prove useful for strategies involving LRAs to purge latently infected cells from HIV-1 infected individuals. IACS-9571 causes cause synergistic reactivation of viral expression in combination with PEP005 (Fig. 3.9), and we suggest this combination may prove useful for implementation of potential therapies involving reactivation of HIV-1 provirus expression. Accordingly, we note this compound was proposed as therapy for glioblastomas where TRIM24 is a contributing factor for malignancy (Han & Sun, 2021), and that PEP005 is presently in clinical trials for skin cancers (Collins et al, 2019). 3.4 Materials and methods 3.4.1 Cell and virus culture Jurkat and HEK293T cells were maintained in culture conditions as described in Chapter 2. VSV-G pseudotyped viruses were produced by co-transfection of HEK293T cells with viral molecular clone psPAX and VSV-G and infections were performed as described in Chapter 2. Human Peripheral Blood CD4+ T cells were purchased from STEMCELL Technologies (Catalog # 200-0165). T cells were cultured in RPMI-1640 media supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), and 30 U/mL IL2, and infection with RGH was performed as previously described (Battivelli & Verdin, 2018). Briefly, cells were first incubated for three days with DynabeadsTM Human T-Activator CD3/CD28 beads. The beads were removed and cells were infected with RGH at a multiplicity of infection (M.O.I.) that resulted in less than 8% of the population infected. 80 Peripheral Blood Mononuclear Cells (PBMC) from participants with HIV-1 on ART were isolated from whole blood by density gradient centrifugation using LymphoprepTM and SepMateTM tubes (StemCell Technologies) and cryopreserved. Upon thawing, PBMCs were cultured in RPMI supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL). Samples from donor participants were collected with informed consent obtained from all subjects and/or their legal guardian, and handled using methods in accordance with guidelines and protocols approved by the University of British Columbia Clinical Research Ethics Board, certificate H16-02474. 3.4.2 Immunoblotting Western blotting was performed as previously described (Hashemi et al, 2018), and unprocessed images are in Fig. S3.5. Antibodies were as follows: Tubulin, Abcam ab7291 (1:10000 – 1:40000); Flag, Sigma Aldrich F3165 (1: 10000); Myc, Santa Cruz sc-40 (1:500 – 1:1000); TRIM24, Proteintech 14208-1-AP (1:2000); Goat Anti-Rabbit-HRP (1:2000000), Abcam ab6721; Goat Anti-Mouse-HRP, Pierce 1858413 (1:20000). 3.4.3 Chromatin Immunoprecipitation ChIP of Jurkat mHIV-Luciferase cells was performed as described in Chapter 2. Antibodies for ChIP were: TRIM24, Proteintech 14208-1-AP (10 μg); RNAPII, Abcam ab26721 (5 μg); RNAPII pS5, Abcam ab5408 (5 μg); RNAPII pS2, Abcam ab238146 (5 μg); CDK9, Abcam ab239364 (10 μg); Flag, Sigma Aldrich F3165 (5 μg); Mouse IgG, Santa Cruz sc-2025 (paired to target antibody); Rabbit IgG, Abcam ab1722730 (paired to target antibody). 3.4.4 Luciferase reporter assays Luciferase reporter assays were performed as described in Chapter 2. 3.4.5 Q-RT-PCR Following the indicated treatment, RNA was extracted from cells using RNeasy Kit (Qiagen) and subsequently analyzed with the Quant Studio 3 Real-Time PCR system (Applied Biosystems) using Power SYBR® Green RNA-to-CT™ 1-Step Kit (Thermo Fisher) as per the manufacturer’s instructions. RT-PCR data was normalized to GAPDH expression using the ΔΔCt method as previously described (Livak & Schmittgen, 2001). Cycling parameters were as follows: 48 oC, 30 min, 1x; 95 oC, 10 min, 1x; 95 oC, 15 sec, 60 oC, 1 min, 40x. Primers are as follows: IL2, Fwd 5’ AACTCACCAGGATGCTCACA, Rev 5’ GCACTTCCTCCAGAGGTTTGA; CD69, Fwd 5’ TCTTTGCATCCGGAGAGTGGA, Rev 5’ ATTACAGCACACAGGACAGGA; CXCL10, Fwd 5’ AAGTGGCATTCAAGGAGTACCT, Rev 5’ GGACAAAATTGGCTTGCAGGA; IFN-γ, Fwd 5’ ATTCGGTAACTGACTTGAATGTCC, Rev 5’ CTCTTCGACCTCGAAACAGC; IL8, Fwd 5’ ACTGAGAGTGATTGAGAGTGGAC, Rev 5’ AACCCTCTGCACCCAGTTTTC; CSF2, Fwd 5’ ACCTGCCTACAGACCCGCCT, Rev 5’ GAAGTTTCCGGGGTTGGAGGGC; CCL22, 81 Fwd 5’ CGCGTGGTGAAACACTTCTAC, Rev 5’ GCCACGGTCATCAGAGTAGG; HIV-1 mRNA, Fwd 5’ CTTAGGCATCTCCTATGGCAGGA, Rev 5’ GGATCTGTCTCTGTCTCTCTCTCCACC; GAPDH, Fwd 5’ TGCACCACCAACTGCTTAGC, Rev 5’ GGCATGGACTGTGGTCATGAG. 3.4.6 Flow cytometry Cells were treated as indicated in the figure legends. Subsequent to the stated treatment, cells were collected, resuspended in PBS, and analyzed on a BD Biosciences LSRII-561 system where threshold forward scatter (FSC) and side scatter (SSC) parameters were set so that only a homogenous population of live cells were counted (Fig. S3.4A). Data was analyzed and mean fluorescent intensities were determined using FlowJo software (TreeStar). For surface staining of CD69, 1x106 mHIV-Luciferase cells were treated for 4 hrs in the presence of DMSO (Ve control), 10 μM IACS-9571, 10 nM PMA, or 10 nM PMA and 10 μM IACS-9571. Following treatment, blocking was performed by adding CD16/CD32 Rat anti-Mouse, Clone: 2.4G2 (BD Biosciences 553141) and incubating on ice for 15 min. Subsequently, PE-Cy™7 Mouse IgG1 κ Isotype Control (BD Biosciences 557872) or PE-Cy™7 Mouse Anti-Human CD69 (BD Biosciences 561928) was added, and sample were incubated 30 min on ice. Samples were then washed 2x with ice cold PBS and resuspended in PBS for analysis on BD Biosciences LSRII-561 system as previously stated. 3.4.7 Quantitative analysis of IACS-9571 and latency reversing agent interaction Bliss independence modeling was used as previously described (Laird et al, 2015) (Liu et al, 2018) to statistically assess the activity of IACS-9571 in combination with various LRAs on HIV-1 expression. The equation Faxy, P = Fax + Fay – (Fax)(Fay) defines the Bliss independence model, in which Faxy, P is the predicted fraction affected by a combination of drug x and drug y that is derived from the experimentally observed fraction affected by drug x (Fax) and drug y (Fay) individually. Comparison of the predicted combinatorial affect (Faxy, P) with the experimentally observed impact (Fa xy,O) was then performed: ∆Fa xy = Fa xy,O − Fa xy,P. If ∆Fa xy is greater than 0, the combination of drugs x and y exceed that of the predicted affect indicating that the drugs display synergistic interaction. If ∆Fa xy = 0, the drug combination follows the Bliss model for independent action. If ∆Fa xy is less than 0, the drug interaction is antagonistic as the observed effect of the drug combination is less than predicted. In this analysis, the fraction affected was calculated as follows: Fax = (HIV expression of drug x at the indicated time – HIV expression at time 0 hrs) / (Max HIV expression observed over the time course – HIV expression at time 0 hrs). 3.4.8 Statistics and reproducibility Details of statistical analysis are indicated in Figure Legends. Mean is shown with standard deviations. Unpaired sample t-tests or ANOVA, as indicated, were performed using GraphPad Prism 9.0.0, and statistical significance is indicated at *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001. 82 3.4.9 Data availability All data pertaining to the findings of Chapter 3 are available within the article Horvath et al, 2023 and its Supplementary Information, or are available from the corresponding author, I. Sadowski, ijs.ubc@gmail.com. 3.5 Chapter acknowledgments We thank Andy Johnson and Justin Wong of the UBC Flow Cytometry Facility for performing FACS analysis as well as for assistance with flow cytometry. We thank the laboratory staff at the BC Centre for Excellence in HIV/AIDS for processing PBMCs from study participants. This research was supported by program project grant F16-01210, from the Canadian Institutes of Health Research (CIHR). PBMC collection from participants with HIV-1 was supported by CIHR project grant PJT-159625 (to ZLB). ZLB is supported by a Michael Smith Health Research BC Scholar Award. 3.6 Supplementary figures Figure S3.1 IACS-9571 promotes HIV-1 expression. A: Schematic representation of HIV-1 virus integrated in the JLat10.6 cell line. The full-length virus expresses GFP in place of Nef with mutations to Env rendering it replication incompetent. B: JLat10.6 cells were treated with IACS-9571 at the indicated concentration for 24 hrs and subsequently analyzed by flow cytometry. Results are the average of two measurements, error bars represent standard deviation. Statistical analysis was performed using unpaired t-test. C: JLat10.6 cell viability was determined Following 24 hrs treatment with the indicated concentration of IACS-9571. Assays were performed in duplicate and error bars depict standard deviation. 83 Figure S3.2 Quantitative analysis of synergy between IACS-9571 and latency reversal agents. A – G: Calculation of synergy between IACS-9571 and the indicated latency reversal agent at the given time point was performed using Bliss Independence Modelling. Data are presented as the difference between the predicted and the observed fractional HIV-1 expression 84 response to the given drug combination. See Materials and Methods for more details. H: As in (A – G) but calculated at 6 hrs post-incubation with the combination of IACS-9571 and the indicated compound. Figure S3.3 Jurkat Tat Clone #11 is responsive to IACS-9571. A: Clone #11 cells were treated with 40 nM PMA or 40 nM PMA and 10 µM IACS-9571. Flow cytometry was performed following 24 hrs incubation. Displayed are the results of duplicate experiments, with error bars representing standard deviation. Statistical analysis was performed using unpaired t-test. B: Viability of Clone #11 cells treated with 40 nM PMA and the indicated concentration of IACS-9571 was determined following 24 hrs incubation. Displayed are the results of two determinations with error bars depicting standard deviation. 85 Figure S3.4 Analysis of surface CD69 by flow cytometry. A: Representative scatter plots depicting the gating strategy employed. Threshold forward scatter (FSC) and side scatter (SSC) settings were set as to isolate a homogeneous population of live cells that were then gated (Left, Lymphocytes). The gated population was then examined for fluorescence of PE-Cy7 (Right). B, C: Representative flow cytometry scatter plots detecting surface associated CD69. Jurkat mHIV-Luciferase cells were treated with a DMSO control (Ve), 10 μM IACS-9571, 20 nM PMA, or 10 μM IACS-9571 in combination with 20 nM PMA. Following treatment, cells were stained with PE-Cy7 conjugated antibodies against IgG (B) or CD69 (C) and analyzed by flow cytometry. 86 Figure S3.5 Full length western blots. 87 Chapter 4: Upstream Stimulatory Factors regulate HIV-1 latency and are required for robust T cell activation 4.1 Introduction Previous work has determined that the USF factors directly bind at least three elements within the HIV-1 LTR; the highly conserved RBE1 (CAGCTG) and RBE3 (ACTGCTGA) elements located at the transcription start site and~ -130 bp respectively (Chen et al, 2005) (Dahabieh et al, 2011), and a less conserved E-Box (CACGTG) positioned at ~ -166 bp (d'Adda di Fagagna et al, 1995) (Estable et al, 1996) (Sieweke et al, 1998). It is believed that the USF factors bind to these elements as heterodimers. Additionally, it is generally thought that the USFs potentiate LTR transcription. However, these studies have relied on transient transfection assays and/or cis-element mutations that abolish interactions of multiple factors including USF1, USF2, TFII-I, and YY1 (Chen et al, 2005) (d'Adda di Fagagna et al, 1995) (Dahabieh et al, 2011) (Du et al, 1993) (Naghavi et al, 2001) (Sieweke et al, 1998). Here, we demonstrate that proviral expression is disrupted upon depletion of USF2 while USF1 is not required. Additionally, loss of USF2 results in the reduction of USF1 protein expression, an effect that does not correlate with USF1 mRNA transcript abundance. Furthermore, we detected that USF1 and USF2 are key factors that facilitate T cell activation as loss of either protein triggered wide-spread constraint of T cell signal induced genes. Additionally, USF2 knockout limited T cell activation to a greater extent than USF1 and manifested a cellular state that was restrictive to HIV-1 infection. These observations delineate USF1 and USF2 mediated regulation of HIV-1 and identify these proteins as key signal regulated transcription factors that play a vital role in activation of the T cell inflammatory response. 4.2 Results 4.2.1 Regulation of HIV-1 expression by USF1 and USF2 The 5’ LTR serves as the HIV-1 promoter and contains several elements that bind USF1/2. The RBE1 and RBE3 elements are both occupied by the RBF-2 complex that is minimally composed of USF1/USF2 and TFII-I, while USF1/USF2 heterodimers bind an additional upstream E-Box (Fig. 4.1A). Previous studies have implicated USF1 and/or USF2 as regulators of HIV-1 expression, mostly based on transient transfections assays and LTR mutations that abolish binding of these factors (d'Adda di Fagagna et al, 1995) (Du et al, 1993) (Naghavi et al, 2001) (Sieweke et al, 1998). To examine specific effects of USF1 and USF2 on HIV-1 expression we produced USF1 and USF2 gene knockouts (KO) (Fig. 4.1C, 4.1D) in the Jurkat Tat mHIV-Luc T cell line bearing an integrated HIV-1 mini-virus where luciferase expression is driven by the 5’ LTR (Fig. 4.1B) (Bernhard et al, 2011) (Horvath et al, 2023) (Horvath et al, 2023). Surprisingly, we noted that USF1 protein abundance was reduced in all USF2 knockout lines (Fig. 4.1D), whereas USF2 protein expression was not affected by USF1 knockout (Fig. 4.1C). We examined activation of HIV-1 expression in these knockout lines and found that USF1 disruption tended to cause a slight increase in luciferase expression in 88 unstimulated as well as activated conditions as mediated by the T cell signaling agonist PMA (Fig. 4.1E). In contrast, provirus expression was diminished in both unstimulated and stimulated conditions in all USF2 knockout lines (Fig. 4.1F). Consistent with results using the knockout lines, we found that that shRNA mediated depletion of USF1 (Fig. S4.1A) had no effect on HIV-1 provirus expression (Fig. S4.1B), while shRNA knockdown of USF2 (Fig. S4.1C) inhibited HIV-1 expression in both untreated cells and cells stimulated with the phorbol ester PMA (Fig. S4.1D). Additionally, TZM-bl cells were refractory to HIV-1 induction upon USF2 depletion (Fig. S4.2), confirming the role of USF2 for expression of chromosomally integrated HIV-1. As USF1 protein expression was dependent upon USF2 in Jurkat T cells, we examined the effect of USF2 gene knockout in HEK293T cells where we also observed loss of both USF1 and USF2 protein expression (Fig. 4.2A). We examined the effect of USF2 knockout in these cells using transfection with an LAI-derived LTR-Tat-IRES-GFP reporter construct (Fig. S4.3A, S4.3B). Consistent with the above results, we observed significantly lower expression of GFP expression in HEK293T USF2 knockout lines transfected with this reporter relative to WT (Fig. 4.2B). Remarkably, we found that co-transfection of HEK293T USF2 KO cells with vectors expressing either USF1 or USF2 (Fig. 4.2C) with the HIV-1 LTR-Tat-IRES-GFP reporter not only rescued LTR transcription but caused activation of GFP expression greater than that of wildtype cells (Fig. 4.1D, S4.3C). These observations suggest that any combination of USF1 and USF2 can activate transcription from the HIV-1 LTR but because USF1 protein expression is significantly decreased in USF2 knockout lines while USF2 expression is not affected in USF1 knockouts, only the USF2 knockouts produce a significant effect on HIV-1 LTR transcription. Of note, knockout of either USF1 or USF2 was not associated with any noticeable change in phenotype and cell growth was unaffected (Fig. S4.4). 89 Figure 4.1 USF2 knockout disrupts HIV-1 expression. A: Depiction of the core promoter and proximal enhancer region of the HIV-1 5’ LTR. RBE1 and RBE3 are bound by RBF-2, a complex minimally composed of USF1/2 and TFII-I. TFII-I directly interacts and recruits the TRIM24 co-factor to the RBE1/3 elements. B: Representation of the Luciferase reporter provirus that is chromosomally integrated in the Jurkat mHIV-Luc cell line. The 5’ LTR drives luciferase expression while an internal EF1α constitutive promoter expresses GFP which was used for cell line selection. C, D: USF1 (A) and USF2 (B) KO cell lines were produced using CRISPR-Cas9 in the Jurkat mHIV-Luciferase parental cell line. Immunoblotting of cellular lysates was performed using antibodies against USF1, USF2, and Tubulin. E: Wildtype or USF1 KO mHIV-Luciferase cells were left untreated (Ve, DMSO) or incubated with 5 nM PMA for 4 hours prior to measuring luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. F: Same as in E, but USF2 KO cell lines were used instead of USF1 KO cells (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 90 Figure 4.2 USF1 and USF2 activate HIV-1 LTR transcription. A: USF2 KO clonal cell lines were generated from the HEK293T parental line through CRISPR-Cas9 gene editing. KO was confirmed by immunoblotting whole cell extracts using antibodies against USF1, USF2, and Tubulin. B: Wildtype and USF2 KO HEK293T cells were co-transfected with LAI derived LTR-Tat-IRES-GFP reporter plasmid and constitutive EF1α-RFP vector (Fig. S4.3A). The GFP mean fluorescent intensity (MFI) of the RFP expressing population was determined one day post-transfection by flow cytometry and normalized to wildtype cells (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: HEK293T USF2 KO cells were transfected with an empty vector (EV) or vectors expressing either USF1 or USF2. Whole cell lysates were examined by immunoblotting with antibodies against USF1, USF2, and Tubulin. D: Wildtype and USF2 KO HEK293T cell lines were co-transfected with LAI LTR-Tat-IRES-GFP reporter plasmid and either CMV-EV-EF1α-RFP (EV), CMV-USF1-EF1α-RFP (USF1) or CMV-USF2-EF1α-RFP (USF2) vector (Fig. S4.3A). One day post-transfection, the GFP mean fluorescent intensity (MFI) of the RFP expressing population was determined by flow cytometry (Fig. S4.3C) and normalized to CMV-EV-EF1α-RFP (EV) transfected wildtype cells (n = 3, mean ± SD). Statistical analysis was performed using one-way ANOVA. 91 4.2.2 Effect of USF factors on HIV-1 activation in response to latency reversing agents and Tat We examined the effect of USF1 and USF2 knockouts on reactivation of HIV-1 in response to various mechanistically diverse latency reversing agents (LRAs). In T cells, the phorbol ester PMA activates protein kinase C (PKC) and Ras-MAPK signaling, while ionomycin causes release of intracellular calcium activating calcineurin, and treatment with this combination mimics T cell activation produced by CD4 receptor engagement (Sadowski & Mitchell, 2005). We found that USF2 KO inhibited HIV-1 provirus activation in response to PMA or ionomycin individually, or in combination (Fig. 4.3A). In contrast USF1 KO caused a slight increase in response to the combination of PMA/ionomycin or PMA alone (Fig. 4.3A). The effects of the USF1 and USF2 knockouts on response to the combination of PMA and ionomycin was most pronounced at 6 hours post treatment (Fig. 4.3B). As observed previously, we found that HIV-1 expression becomes repressed upon prolonged treatment (20 hrs) with PMA/ ionomycin in WT cells (Malcolm et al, 2007), and this effect was not significantly different in the USF1 and USF2 knockout lines (Fig. 4.3B). Interestingly, loss of USF1 caused slightly decreased induction following treatment with ionomycin alone (Fig. 4.3A), indicating that USF1 is not required for HIV-1 reactivation in response to PKC and Ras-MAPK signaling but may play a role for activation by calcium-calcineurin signaling. Ingenol 3-angelate (PEP005) also activates PKC signaling (Kedei et al, 2004), and similar to the results with PMA, we observed a significantly lower response to PEP005 in USF2 KO cells, but no effect in the USF1 KO line (Fig. 4.3A). These results indicate that USF2 is required for reactivation of HIV-1 in response to PKC, Ras-MAPK, and calcineurin T cell signaling pathways, whereas USF1 may cause some inhibition of response to MAPK activation. Latent provirus can be reactivated independent of T cell signaling by altering the epigenetic composition or interaction of bromodomain containing proteins with the LTR. Treatment with the histone deacetylase inhibitor (HDACi) suberanilohydroxamic acid (Desimio et al, 2017) modestly increased HIV-1 expression, an effect that was limited by both the USF1 and USF2 knockouts (Fig. 4.3A), indicating that these factors regulate HIV-1 latency through organization of LTR chromatin structure involving histone acetylation. Next, we examined the effect of the USFs on reactivation in response to the bromodomain inhibitors JQ1 and IACS-9571. JQ1 is a well-studied LRA that inhibits the BRD4 bromodomain causing increased Tat transactivation (Li et al, 2013), while IACS-9571 inhibits the TRIM24 bromodomain causing increased LTR occupancy of this factor and stimulation of transcriptional elongation (Horvath et al,2023) (Horvath et al, 2023). We found that the USF2 knockout significantly limited the effect of JQ1, whereas USF1 KO had no effect (Fig. 4.3A). In contrast, the USF2 knockout, and to a lesser extent USF1 KO inhibited reactivation of HIV-1 expression in cells treated with IACS-9571 (Fig. 4.3A), suggesting that both USFs impact regulation of HIV-1 by TRIM24. We also examined the effect of USF2 gene knockout on activation of HIV-1 expression by the viral transactivator protein Tat. For this experiment we transfected USF2 knockout 92 HEK293T lines (Fig. 4.2A) with HIV-1 LTR-Tat-IRES-GFP (+Tat), or LTR-GFP reporter plasmids (-Tat) (Fig. S4.5A, S4.5B). We observed that GFP expression from both HIV-1 LTR reporter plasmids was significantly lower in the USF2 null cells compared to WT (Fig. 4.3C, S4.5C, S4.5D). These observations indicate that full activation of HIV-1 transcription by Tat requires USF2 protein, although USF2 also has a significant effect on HIV-1 expression in the absence of Tat. 93 Figure 4.3 Role of USF factors for the induction of HIV-1 in response to latency reversal agents and Tat. A: Wildtype, USF1 KO, or USF2 KO mHIV-Luciferase cells were left untreated or treated with 5 nM PMA/ 1 μM ionomycin, 5 nM PMA, 10 nM PEP005, 1 μM ionomycin, 10 μM SAHA, 10 μM JQ1, or 10 μM IACS-9571 for 6 hrs prior to luciferase assay (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. B: Wildtype, USF1 KO, or USF2 KO mHIV-Luciferase were treated with 5 nM PMA and 1 μM ionomycin for the indicated amount of time, at which point luciferase expression was measured (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: Wildtype or USF2 KO HEK293T cell lines were co-transfected with a EF1α-RFP vector and either an LTR-GFP (-Tat) or LTR-Tat-IRES-GFP (+Tat) reporter construct (Fig. S5A, S5B). One day post-transfection, the mean fluorescent intensity (MFI) of GFP for the RFP transfected population was determined by flow cytometry (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 4.2.3 USF2 stabilizes USF1 protein Previously, we observed that depletion of USF2 in Jurkat T cells (Fig. 4.1D, S4.1C) or HEK293T cells (Fig. 4.2A) invariably caused a corresponding decrease in USF1 protein abundance. To examine the mechanism by which USF1 protein abundance is dependent upon USF2, we first generated a Jurkat USF2 KO cell line where Flag tagged USF2 is expressed from a doxycycline-inducible promoter. We found that expression of USF2-Flag in the USF2 KO cell line, upon addition of doxycycline, rescued expression of USF1 protein to greater levels as compared to WT cells (Fig. 4.4A). We also observed that USF2 KO did not cause a decrease in USF1 mRNA abundance as compared to WT cells (Fig. 4.4B), regardless of T cell activation state (Fig. 4.4C), indicating that differences in USF1 expression in USF2 KO cells must relate to protein stability or effects on mRNA translation. Finally, PMA/ionomycin induced T cell activation did not alter USF1 or USF2 protein abundance in WT cells, and USF1 protein levels were decreased in USF2 knockout cells regardless of cellular activation state (Fig. 4.4D). 94 Figure 4.4 USF1 expression is stabilized in the presence of USF2. A: USF2 KO Jurkat T cells were transduced with doxycycline (Dox) inducible Flag tagged USF2 expression vector. Following incubation with 1 μg/mL Dox for the indicated time point, whole cell lysates were collected and subject to immunoblotting using antibodies targeting USF1, USF2, and Tubulin. B: RNA was extracted from wildtype and USF2 KO Jurkat cells and subject to RT-PCR using primers targeting USF2 mRNA. USF2 transcript expression was normalized to GAPDH (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: Normalized RNA-seq USF1 mRNA counts of wildtype and USF2 KO Jurkat cells under basal (Ve) and T cell activated condition (PMA/Ion) (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. D: Wildtype, USF1 KO, or USF2 KO cells were left untreated (Ve, DMSO) or stimulated by treatment with 5 nM PMA and 1 μM ionomycin. Following 4 hrs, whole lysate was collected and immunoblotted using antibodies against USF1, USF2, and Tubulin. 95 4.2.4 HIV-1 LTR binding of USF proteins We examined the requirements of USF1 and USF2 for binding to the HIV-1 LTR using chromatin immunoprecipitation and analysis by quantitative PCR (ChIP-qPCR) (Fig. 4.5A). Because we were unable to immunoprecipitate USF1 with antibodies against native protein, we generated mHIV-Luc Jurkat T USF1 and USF2 KO cell lines that stably express Myc tagged USF1 (Fig. 4.5B). We found that USF1-Myc was associated with the LTR expressed in both the USF1 and USF2 KO cell lines (Fig. 4.5C). However, we did observe slightly higher amounts associated with all three sites on the LTR in the USF1 knockout line, which expresses endogenous USF2 (Fig. 4.5C). Similarly, we found that USF2 was associated with the LTR at equivalent amounts in WT and the USF1 knockout lines (Fig. 4.5D). Furthermore, PMA/ionomycin mediated T cell activation had no effect on USF2 binding of RBE3 or RBE1 (Fig. 4.5E), consistent with the observation that TFII-I is constitutively bound to the HIV-1 LTR (Horvath et al, 2023). These results indicate that USF1 and USF2 are both capable of binding specific cis-elements on the LTR as homodimers in vivo, a result that is consistent with binding of recombinant USF1 and USF2 proteins in vitro (Malcolm et al, 2008) (Chen et al, 2005). Thus, although USF1 and USF2 were predominately shown to function as heterodimers in vivo (Sirito et al, 1994) (Sawadogo et al, 1988), USF2 is capable of binding independently of USF1 at multiple sites in the HIV-1 LTR for activation of transcription. In contrast, as indicated above, USF1 protein abundance is dependent upon USF2, and therefore is incapable of regulating HIV-1 transcription in USF2 knockout cells. Next, we sought to determine the factor requirement for binding of the RBF-2 complex to RBE3/1 elements. To this end we performed shRNA mediated knockdown of TFII-I (GTF2I) (Fig. 4.6A) in the Jurkat mHIV-Luc cell line (Fig. 4.1B). Consistent with previous results (Horvath et al, 2023), we observed that proviral expression was limited upon TFII-I depletion (Fig. 4.6B), an effect similar to USF2 KO (Fig. 4.1F). Interestingly, we observed no change in binding of USF2 to the RBE elements upon TFII-I knockdown (Fig. 4.6C). In contrast, binding of TFII-I was abrogated upon knockout of USF2 (Fig. 4.6D). TRIM24 is a co-activator of HIV-1 expression that requires TFII-I for LTR recruitment (Horvath et al, 2023). In agreement with the binding pattern of TFII-I, we found that TRIM24 was associated with the LTR only in USF2 expressing cells (Fig. 4.6E). Collectively, these results indicate that USF factors are required for RBF-2 complex formation. . 96 Figure 4.5 USF1 and USF2 are capable of localizing independently to the LTR. A: Schematic representation of the primer sites that were used for ChIP-qPCR analysis. B: USF1 or USF2 knockout cells were transduced with an empty vector (EV) or Myc tagged USF1 expression vector. Whole cell lysates were collected and immunoblotted with antibodies against Myc and Tubulin. C: USF1 or USF2 deleted Jurkat T cells transduced with empty vector (EV) or USF1-Myc expression vector were subject to ChIP-qPCR using antibodies against Myc. ChIP-qPCR results are normalized by subtraction of values produced with sample paired non-specific IgG immunoprecipitation (n = 2, mean ± SD). Statistical analysis was performed using two-way ANOVA. D: Wildtype or cells possessing USF1 or USF2 genetic deletions were subject to ChIP-qPCR using antibodies targeting endogenous USF2. ChIP-qPCR results are normalized by subtraction of values produced with sample paired non-specific IgG immunoprecipitation (n = 2 - 3, mean ± SD). Statistical analysis was performed using two-way ANOVA. E: Jurkat mHIV-Luc cells were incubated with a vehicle control (Ve, DMSO) or 5 nM PMA/ 1 μM ionomycin for 4 hrs after which ChIP-qPCR was performed using antibodies against USF2. Shown are qPCR 97 replicates determined by subtraction of values produced by sample paired non- specific IgG immunoprecipitation (n = 2 - 3, mean ± SD). Statistical analysis was performed using unpaired t-test. Figure 4.6 Factor determinants of RBF-2 formation. A: Jurkat mHIV-Luc cells were transduced with an LKO control shRNA or a TFII-I targeting shRNA vector. Transduced cells were selected for with 7.5 μg/mL puromycin for 8 days after which lysates were analyzed by immunoblotting using antibodies targeting TFII-I and Tubulin. B: shRNA transduced mHIV-Luc cells were left unstimulated (Ve, DMSO) or stimulated with 5 nM PMA for 4 hours prior to assessing luciferase expression (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: Jurkat mHIV-Luc cells were transduced with the indicated shRNA, selected with puromycin, and subject to ChIP-qPCR using antibodies against USF2. Depicted are the results of qPCR performed in replicate, determined by subtraction of values produced by sample paired non- specific IgG immunoprecipitation (n = 3, mean ± SD). D: Wildtype of USF2 KO mHIV-Luc cells were subject to qPCR with antibodies targeting TFII-I. Replicate qPCR results were determined by subtraction of values produced by sample paired non- specific IgG immunoprecipitation (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. E: As in D, but ChIP was performed using antibodies against TRIM24 (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 98 4.2.5 Global regulation of transcription by USF1 and USF2 in unstimulated T cells Given the surprising observation that Jurkat T cells with USF1 and USF2 gene knockouts have distinct effects on HIV-1 expression, we sought to determine the degree that these factors cooperatively regulate global gene expression. To examine these effects, we performed RNA-seq on cell lines bearing USF1 or USF2 gene knockouts. In this analysis we identified 236 differentially expressed genes (DEG) upon USF1 depletion and 563 DEG following USF2 disruption (Fig. 4.7A, 4.7B). Of the genes affected by loss of USF1, slightly more were down-regulated than up-regulated (146 to 90, respectively), while USF2 depletion primarily resulted in decreased expression of target genes (410 down-regulated to 153 up-regulated) (Fig. 4.7A, 4.7B). Comparison of transcriptomes from USF1 and USF2 KO cells demonstrated some overlap amongst down-regulated genes with 34% of USF1 KO down-regulated genes similarly regulated in USF2 KO cells (Fig. 4.7C). However, we observed only 7% of genes upregulated upon USF1 depletion being likewise upregulated in USF2 knockout cells (Fig. 4.7D). Overall, therefore the USF1 and USF2 knockouts cause a surprisingly small overlap in differential gene regulatory effects in unstimulated Jurkat T cells (Fig 7E). Furthermore, Gene Set Enrichment Analysis (GSEA) indicated only one pathway with less than 25% false discovery rate (FDR) shared between USF1/2 KO, “TNFA signaling via NFkB”, which was negatively enriched (Fig. 4.7F, 4.7G). These results indicate that USF1 and USF2 share regulation for a minor subset of genes in unstimulated T cells. 99 Figure 4.7 Transcriptomic effects of USF1 or USF2 knockout under basal conditions. A, B: Volcano plots depicting DESeq2 analysis comparing Wildtype and USF1 KO (A) or USF2 KO (B) Jurkat T cells. Analysis was performed on n = 3 RNA-seq samples with significant genes possessing p-value < 0.05 and fold change in expression > 1.5. C, D: Venn diagram depiction of genes significantly down- (C) or up-regulated (D) upon USF1 or USF2 KO. E: Heatmap depiction of the change in mRNA expression upon USF1 or USF2 KO of genes that were found to be differentially expressed upon USF1 KO. F, G: Gene set enrichment (GSEA) of Hallmark gene sets that are down-regulated (F) or up-regulated (G) for USF1 or USF2 KO transcriptomes. 100 4.2.6 USF1 and USF2 cooperatively regulate global gene expression in activated T cells USF1 and USF2 are signal regulated transcription factors (Galibert et al, 1997) (Horbach et al, 2015) (Nowak et al, 2005) (Spohrer et al. 2017) that are phosphorylated upon T cell activation (Chen et al, 2005). Consequently, we also examined global gene expression in USF1 and USF2 knockout lines using RNA-seq from cells stimulated with PMA and ionomycin co-treatment. This analysis revealed a greater effect on global expression overall in stimulated cells compared to unstimulated, with 1846 and 2667 DEG upon loss of USF1 and USF2, respectively (Fig. 4.8A, 4.8B). Importantly, depletion of either USF1 or USF2 resulted in the majority of DEG having decreased expression rather than increased (Fig. 4.8A, 4.8B). These results support the contention that USF1 and USF2 are T cell signal regulated factors that preferentially behave as transcriptional activators. Additionally, we observed a significantly greater proportion of genes that were similarly affected by USF1 KO and USF2 KO in activated T cells. In stimulated cells, we found that 68% of USF1 KO downregulated and 38% of USF1 KO upregulated genes were similarly mis-regulated upon USF2 KO (Fig. 4.8C, 4.8D). Furthermore, a minority of USF1 DEG were found to be mis-regulated opposite that of USF2 null cells (Fig. 4.8E). We performed GSEA analysis from USF1 KO and USF2 KO T cell transcriptomes to determine the predominant affected pathways, which revealed effects on genes involving “TNFA Signaling via NFkB”, “Hypoxia”, and “Interferon Gamma Response” being negatively enriched (Fig. 4.8F), while “Oxidative Phosphorylation” and “Myc Targets V1” were found to be positively enriched in both USF1 KO and USF2 KO cell lines (Fig. 4.8G). Although genes from multiple pathways were similarly enriched in the USF1 and USF2 KO lines, we observed asymmetrical regulation between these factors as pathways including “mTORC1” and “Notch Signaling” were negatively enriched solely in USF1 and USF2 knockout cells, respectively (Fig. 4.8F). Interestingly, USF1 was previously implicated in regulating the UV and tanning response (Galibert, 2001) (Corre et al, 2004) (Bouafia et al, 2014), yet we found that “UV Response Down” was negatively enriched in cells deficient of either USF1 or USF2 (Fig. 4.8F) suggesting that both factors mediate this process. Taken together, these results indicate that USF1 and USF2 display a high degree of cooperativity for global gene regulation during T cell activation. As noted above, loss of USF2 results in a corresponding decrease in USF1 expression, and therefore it is possible that USF2 may partially compensate for loss of USF1 protein in both unstimulated and stimulated T cells. 101 Figure 4.8 Regulation of the activated T cell transcriptome by USF1 and USF2. A, B: Volcano plots depicting DESeq2 analysis comparing Wildtype and USF1 KO (A) or USF2 KO activated T cells (B). Jurkat T cells were stimulated by PMA/ionomycin co-treatment for 4 hrs prior to RNA extraction. Analysis was performed on n = 3 RNA-seq samples with significant genes possessing p-value < 0.05 and fold change in expression > 1.5. C, D: Venn diagrams displaying genes significantly down- (C) or up-regulated (D) upon knockout of USF1 or USF2 under activating conditions. E Heatmap depiction of the change in gene expression upon USF1 102 or USF2 KO of genes that were differentially expressed upon USF1 KO under activated T cell conditions. F, G: Gene set enrichment (GSEA) of Hallmark gene sets that are down-regulated (F) or up-regulated (G) for USF1/ USF2 KO transcriptomes following PMA/ionomycin mediated T cell activation. 4.2.7 USF1 and USF2 facilitate T cell activation Having observed that loss of either USF1 or USF2 generated an abundance of down-regulated genes upon T cell activation compared to wildtype, we examined the potential role of these factors in regulating inflammatory response. From RNA-seq analysis we identified 2790 down-regulated and 3481 up-regulated genes in response to T cell activation (Fig. 4.9A). Notably, genes that were induced in response to stimulation displayed a much greater range of expression compared to genes that were inhibited (Fig. 4.9A). We then examined the effect of USF1 and USF2 gene knockout on expression of genes that are activated or repressed upon T cell stimulation. Here we found that 755 (22%) and 1114 (32%) of genes induced upon T cell stimulation showed significantly less expression in the absence of USF1 and USF2 respectively (Fig. 4.9B). Genes that were repressed upon T cell activation were much less affected by USF disruption, with only 187 (7%) and 272 (10%) displaying greater expression upon loss of USF1 or USF2, respectively (Fig. 4.9B). These results are consistent with the above observations indicating the role of USF1 and USF2 as transcriptional activators. T cell activation was previously shown to alter the expression of numerous genes with a wide degree of variance (Tong et al, 2016), consistent with our results indicating that nearly 50% of genes induced upon stimulation showing less than a 3-fold change in expression (Fig. 4.9C). Consequently, it is possible that genes displaying the large variance in induction are regulated by different mechanism(s). To examine this with respect to USF1 and USF2, we segmented genes induced upon T cell activation into three groups by level of induction, designated Low (fold change in expression between 1.5 and 3), Mid (fold change in expression between 3 and 8), and High (fold change greater than 8) (Fig. 4.9C). By stratifying genes based on level of induction, it became apparent that genes that were highly expressed in response to T cell stimulation were more potently inhibited in USF1 or USF2 KO cells (Fig. 4.9D). Furthermore, we found that more of the genes that were down regulated upon USF1 or USF2 depletion are those that are highly induced in response to T cell activation (Fig. 4.9E). Additionally, we found that USF1 and USF2 knockout cells displayed partially defective activation of genes downstream of key T cell signaling pathways including TNFα and Ras (Fig. S4.6). Collectively, these results indicate that USF1 and USF2 are signal regulated factors required for full induction of genes upon T cell activation, a finding that is consistent with the evolved capability of the HIV-1 LTR to pirate transcription factors downstream of T cell signaling (Sadowski & Mitchell, 2005) 103 104 Figure 4.9 USF1 and USF2 facilitate T cell activation. A: Volcano plot depiction of DESeq2 analysis comparing the transcriptomes of unstimulated and PMA/ionomycin activated Jurkat T cells. Analysis was performed on n = 3 RNA-seq samples with significant genes having p-value < 0.05 and fold change > 1.5. B: Pie charts depicting the effects of knockout of USF1 (left) or USF2 (right) on T cell activation induced (top) or repressed (bottom) genes. C: The number of T cell activation genes that are induced, shown as a percent of the total, given the fold change in expression. D: Heatmap depicting the effects of USF1 and USF2 KO on T cell activation induced genes. The T cell activation induced genes are stratified based on expression levels into Low, Mid, and High. E: Pie charts depicting the effects of USF1 or USF2 KO on T cell activation genes that are induced at Low, Mid, and High expression levels. 4.2.8 USF2 deficient cells are refractory towards HIV-1 infection Infection of resting CD4+ T cells by HIV-1 is a highly inefficient process as compared to that of activated cells (Korin & Zack, 1998) (Plesa et al, 2007) (Stevenson et al, 1990) (Swiggard et al, 2005) (Unutmaz et al, 1999) (Zack et al, 1992), and although Jurkat cells proceed through the cell cycle, they too are preferentially infected based on activation state (Dahabieh et al, 2014). Because HIV-1 favors infection of stimulated T cells while loss of USF1 and USF2 dampens T cell activation, we sought to examine the role of these factors for HIV-1 infection. For this purpose, we created USF1 and USF2 knockouts in Jurkat T cells that did not contain a previously integrated provirus. Consistent with the results shown above, we observe loss of USF1 expression in both USF1 and USF2 knockout lines (Fig. 4.10A). These lines were infected with Red-Green-HIV-1 (RGH) (Dahabieh et al, 2013) or HIVGKO (Battivelli & Verdin, 2018) where GFP is expressed from the 5’ LTR and constitutive promoters express mCherry or mKO2 in RGH and HIVGKO, respectively (Fig. 4.10B). These dual reporter HIV-1 derivatives allow differentiation of productive and latent infections by flow cytometry (Fig. 4.10C, 4.10D) (Dahabieh et al, 2014). We found that infection of USF1 knockout cells produced a small but not significant decrease in RGH (Fig. 4.10E) or HIVGKO (Fig. 4.10F) infectivity compared to WT, 4 days post-infection. In contrast, USF2 knockout cells were infected roughly half that of wildtype cells by both reporter virus (Fig. 4.10C-F). Interestingly, although USF2 is required for reactivation of latent provirus, we found no change in the proportion of cells that establish immediate latency upon infection of USF2 KO T cells (Fig. 4.10G, 4.10H). Additionally, infection of USF knockout Jurkat Tat mHIV-Luc cell lines (Fig. 4.1C, 4.1D) with Red-Blue-HIV-1 (RBH) similarly showed that USF2 deficiency causes cells to be resistant to infection (Fig. S4.7). Taken together, these observations indicate that USF2 is important for infection of T cells by HIV-1 by a process that likely involves cellular activation state. 105 106 Figure 4.10 T cells deficient of USF2 are resistant to HIV-1 infection. A: CRISPR-Cas9 was utilized to produce USF1 or USF2 knockout in Jurkat T cells. Whole cell lysates were subject to immunoblotting with antibodies against USF1, USF2, or Tubulin. B: Schematic representation of the HIV-1 reporter viruses used and the assay. Wildtype, USF1 KO, or USF2 KO Jurkat cells were infected with Red-Green-HIV-1 (RGH) or HIVGKO dual-reporter viruses. Four days post- infection, proviral expression was determined by flow cytometry. C, D: Representative scatter plots of RGH (C) and HIVGKO (D) infected cells, four days post-infection. Uninfected cells that are negative for fluorescence are in Q4, latent infections are located in Q1, productive infections are in Q2, and noise produced from viral recombination events are shown in Q3. E, F: The relative infectivity was determined by flow cytometry four days post-infection of Wildtype, USF1 KO, or USF2 KO Jurkat cells with equivalent amounts of RGH (E) or HIVGKO (F) virions (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. G, H: As in E, F but the ratio of productive infections was analyzed by flow cytometry (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 4.2.9 USF2 mediates the establishment of latency upon HIV-1 infection We found that USF2 knockout inhibits reactivation of latent HIV-1 provirus (Fig. 4.1) but does not affect the latency ratio at 4 days post-infection (Fig. 4.10G, 4.10H). To further examine the role of USF for establishment of latency, we monitored the proportion of productively infected cells for three weeks following infection. Previous observations indicate that the internal constitutively expressed mCherry and mKO2 reporters in cells infected with RGH and HIVGKO become silenced following extended culture (Dahabieh et al, 2013) (Battivelli & Verdin, 2018). Consequently, we isolated infected populations by fluorescent activated cell sorting (FACS) two days post infection (Fig. 4.11A), and then WT, USF1 KO, and USF2 KO infected cells were analyzed by flow cytometry over the following 22 days (Fig. 4.11B, 4.11C). Similar to the results with unsorted infected cells (Fig. 4.10), we found that WT, USF1, and USF2 deficient cells produced a nearly equivalent proportion of productively infected cells at 3- and 4-days post-infection (Fig. 4.11D, 4.11E). However, after 4-days infection, USF2 knockout cells accumulated latent HIV-1 infections at an accelerated rate compared to wildtype or USF1 KO cells (Fig. 4.11D, 4.11E). These results indicate that USF2 promotes the expression of HIV-1 and encourages productive replication upon infection. 107 Figure 4.11 USF2 knockout promotes the establishment of latency upon HIV-1 infection. A: Schematic representation of the HIV-1 reporter assay. Wildtype, USF1 KO, or USF2 KO Jurkat cells were infected with RGH or HIVGKO dual-reporter virus at an M.O.I. of ~0.1. Two 108 days post-infection, all latent and productive infections were isolated by fluorescent activated cell sorting (FACS). HIV-1 expression of the infected population was examined over a 22-day time course by flow cytometry. B, C: Representative flow cytometry scatter plot of RGH (B) or HIVGKO (C) infected Jurkat cells at 4- and 10-days post-infection. D, E: Summary of the percent of productive RGH (D) or HIVGKO (E) infections as determined by flow cytometry at each of the indicated time points (n = 2, mean ± SD). 4.3 Discussion The upstream stimulatory factor (USF) was among the first protein factors identified as a sequence specific DNA factor required for activation of transcription in eukaryotes (Sawadogo, 1985) (Gregor et al, 1990), and among the first human factors discovered to bind the 5’ HIV-1 LTR (Du et al, 1993). Various previous observations have indicated that USF1 and USF2 regulate transcription in response to multiple signaling pathways involving protein kinases, including at least MAPK/ ERKs (Corre et al, 2004) (Horbach et al, 2015), Casein kinase 2 (CK2) (Spohrer et al, 2017), Cdk2 (Cheung et al, 1999), protein kinase C (PKC) (Xiao et al, 2002), DNA protein kinase (DNAPK) (Wong et al, 2009), protein kinase A (PKA) (Sayasith et al, 2005), and Glycogen synthase kinase-3 (GSK-3) (Terragni et al, 2011). These protein kinases represent a significant fraction of signaling networks in human cells, which implicates USF1 and USF2 as key regulatory factors involved in coordinating gene expression in response to diverse cellular stimuli. USF1 and USF2 are ubiquitously expressed in human cells (Sirito et al. 1994), and consequently regulation of their function is likely cell type specific. USF1/2 was found to bind the HIV-1 LTR at an E-box element in the upstream modulatory region at -160 to -165 from the transcriptional start site (Maekawa et al. 1991), where it was shown to activate transcription in cooperation with Ets-1 (Sieweke, 1998). The upstream E-box site for USF is not particularly well conserved on LTRs from infected individuals who develop symptoms of AIDS (Estable et al, 1996). USF1 and USF2 were subsequently found to form part of the factor RBF-2, which binds two highly conserved cis-elements at -130 (RBE3) and -20 (RBE1) (Bell & Sadowski, 1996) (Dahabieh et al, 2011). These sites were initially identified because of their requirement for activation of HIV-1 transcription by activated RAS in T cells (Bell & Sadowski, 1996); this factor was purified Jurkat nuclear extracts using oligonucleotide affinity (Estable et al, 1999), and found to contain USF1, USF2 and TFII-I (Chen et al, 2005). The RBE3 element is not typical of other characterized binding sites for USF1/2 (ACTGCTGA), and interaction of USF with this site in vitro and in vivo is dependent upon TFII-I (Malcolm et al, 2008) (Chen et al, 2005). In contrast RBE1 (CAGCTGC) (Bell & Sadowski, 1996), near the transcriptional start, site resembles a typical E-box element (CANNTG) , and consequently binding of USF1/2 to this site is less dependent upon TFII-I (Dahabieh et al, 2011). Consistent with our previous observations regarding RBE3/1 elements and RBF-2, we find that disruption of USF2 inhibits expression of HIV-1 in response to T cell signaling as well as a variety of 109 latency reversing agents (Fig. 4.1-4.3). Furthermore, USF2 disruption inhibits the initial incidence of HIV-1 infection (Fig. 4.10) and reduces the proportion of infected cells that undergo productive gene expression (Fig. 4.11). Mutation of RBE3 and/or RBE1 prevents induction of proviral expression in response to T cell signaling, and although USF1/2 and TFII-1 have been implicated in regulation of signal responsive gene expression (Horbach et al, 2015) (Segura-Puimedon et al, 2013), the mechanism(s) by which RBF-2 activates HIV-1 expression in response to T cell receptor engagement has not been elucidated. TFII-1, in association with serum response factor (SRF) was shown to regulate expression of the c-fos promoter in response to Ras-stimulated MAPK signaling by direct interaction with ERK1 (Kim & Cochran, 2000). Stimulation of T cells by antigen presentation causes activation of multiple pathways downstream of the T cell receptor, including the Ras-Raf-Mek-Erk (MAPK) pathway (Malcolm et al, 2007) (Sadowski & Mitchell, 2005), and consequently the role of USF1/2 for activation of HIV-1 transcription likely includes, at least in part, interaction with TFII-I for binding of the conserved RBE1 and RBE3 sites on the LTR (Malcolm et al, 2008). Considering that USF1 and USF2 are reported to be regulated by multiple protein kinases as described above, it seems likely that these proteins mediate additional function(s) to regulate HIV-1 activation in response to T cell stimulation. Accordingly, we have found that USF1, USF2 and TFII-I all become hyperphosphorylated in T cells stimulated with PMA and ionomycin (Chen et al, 2005). Our RNA-seq analysis found that USF1 and USF2 gene knockouts produce the most significant effect in cells stimulated with PMA and ionomycin, where we observe 1319 and 1977 downregulated genes, respectively (Fig. 4.8), whereas the knockouts have a significantly smaller effect in unstimulated cells (Fig. 4.7). This observation indicates that these factors play a significant role in regulating T cell activation response. Consistently, we observed that a substantial proportion of genes that are highly induced in response to T cell activation were among those most seriously affected by disruption of USF1 or USF2 (Fig. 4.9). Interestingly the most predominant classes of genes that were negatively impacted by USF1 and USF2 knockout in stimulated cells, as identified by gene set enrichment analysis, included those involved in TNFa signaling through NFkB, signaling through Ras, and response to interferon γ (Fig. 4.8F, S4.6) indicating a key role in regulation of inflammatory response. Of note, because the USF factors seem to be ubiquitously expressed (Sirito et al, 1994), and their function is modified by multiple protein kinases, a vast spectrum of cell type specific extracellular responses may be transmitted by these proteins. Despite that USF1 and USF2 are thought to predominately exist as heterodimers in most cell types (Sirito et al, 1998) (Sirito et al, 1994), we find that disruption of their genes cause significantly different alterations in the transcriptome, but where we observed greater overlap between differentially regulated genes in stimulated cells relative to unstimulated (Fig. 4.7, 4.8). It is important to note that we observed distinctly factor specific effects on genes involved in mTORC1 and Notch signaling in activated T cells (Fig. 4.8F). These results are consistent with 110 previous observations indicating distinct functions for USF1 and USF2. For example, USF2 is associated with progression of leukemia through upregulation of HOXA9 but disruption of USF1 alone has minimal effects (Zhang et al, 2020). Additionally, overexpression of USF2 is linked to rheumatoid arthritis that is refractory to treatment through the upregulation of proinflammatory cytokine expression (Hu et al, 2020) while stabilization of USF2 protein by CDK5 dependent phosphorylation promotes prostate cancer proliferation (Chi et al, 2019). Interestingly, differential binding of USF1 and USF2 to the plasminogen activator inhibitor type-1 promoter were reported to regulate expression during G0 to G1 cell cycle transition (Olave et al, 2010) (Qi et al, 2014). The C-terminal DNA binding region of USF1 and USF2 share ~70% sequence identity, however, the N-terminal domains of these proteins display limited sequence homology (Sirito et al, 1998) (Sirito et al, 1994). Consequently, because these factors are capable of binding the same E-box element (Sawadogo et al, 1988), alone or as heterodimers, the specific function of these factors has not been fully characterized. Our results indicate that USF2 is required for USF1 protein expression and accordingly this complicates previous analysis of differential effects of USF1 and USF2 for expression of specific genes. Considering this, it is remarkable that USF1 and USF2 gene knockouts produce notably different transcriptome phenotypes. We find that USF1 protein levels are significantly reduced upon USF2 knockout or shRNA knockdown in Jurkat T cells and HEK293T cells. USF2 depletion does not diminish USF1 mRNA expression in either unstimulated or stimulated T cells (Fig. 4.4), and consequently loss of USF2 must cause USF1 protein instability or inhibit translation of its mRNA. Re-introduction of Flag tagged USF2 in USF2 null cells restored USF1 protein levels (Fig. 4.4A), confirming that the presence of USF2 enables USF1 mRNA utilization or stability. We propose that because USF1 and USF2 predominately exist as heterodimers (Sirito et al, 1994), this interaction stabilizes USF1 protein, and consequently loss of USF2 would result in USF1 protein instability. We have tried to examine this possibility, but this analysis was complicated by the finding that USF1 stability was not enhanced by the proteasome inhibitor MG132, or expression of the C-terminal region of USF2 containing the b-HLH leucine zipper motif. Nevertheless, such a mechanism regulating stability of heterodimer partners has been described previously, for the yeast MATa and a2 proteins are mutually stabilized by their heterodimer formation (Johnson et al, 1998). Our results have revealed novel properties of the ubiquitously expressed USF2 protein and identified a central role for regulation of HIV-1 gene expression. Aberrant expression of this factor was previously linked to additional human diseases, including rheumatoid arthritis (Hu et al, 2020), and cancer (Horbach et al, 2015). Consequently, a more detailed understanding of USF2 function and regulation will provide significant insight towards mechanisms that sculpt the transcriptome in response to extracellular signaling. It will be particularly important to understand the role of USF2 for epigenetic regulation and chromatin organization at the HIV-1 111 LTR and cellular target genes, as well as the relative contribution of associated factors, including TFII-I for signal responsive gene expression. 4.4 Materials and methods 4.4.1 Cell and virus culture Jurkat cell lines and HEK293T derived cell lines were maintained in culture conditions as described in Chapter 2. VSV-G pseudotyped viruses were produced by co-transfection of HEK293T cells with viral molecular clone psPAX and VSV-G and infections were performed as described in Chapter 2. 4.4.2 Immunoblotting Immunoblotting was performed as in Chapter 2. Antibodies were as follows: Tubulin (1:20000) – Abcam ab7291, Flag (1:20000) – Sigma Aldrich F3165, Myc (1:2500) – Santa Cruz Biotechnology sc-40, USF1 (1:500) – Santa Cruz Biotechnology sc-390027, USF2 (1:8000) – Abcam ab264330, Goat Anti-Rabbit-HRP – Abcam ab6721 (1:2000000), Goat Anti-Mouse-HRP – Pierce 1858413 (1:20000). 4.4.3 Luciferase expression assays For Jurkat Tat mHIV-Luciferase expression assays, 1x105 of the luciferase expressing cells were plated with 100 µL media in 96-well plates. Luciferase activity was measured after the indicated time of treatment. Measurements were performed using SuperlightTM luciferase reporter Gene Assay Kit (BioAssay Systems) as per the manufacturer’s instructions; 96 well plates were read in a VictorTM X3 Multilabel Plate Reader. 4.4.4 Transfection assays 6.67x105 Wildtype or USF2 KO HEK293T cells were plated in 2 mL DMEM in 6-well plates. The following day, cells were transfected with 0.5 ng of the indicated LAI LTR-GFP reporter construct and 1 μg of the indicated EF1α-RFP construct. Transfections were performed with polyethylenimine (PEI) at a ratio of 1:4 (DNA:PEI) in Gibco Opti-MEMtm. One day post-transfection, cells were detached with trypsin-EDTA and analyzed by flow cytometry. 4.4.5 Flow cytometry Cells were treated as indicated in the figure legends. Following the indicated treatment, Jurkat derived cells were suspended in PBS while HEK293T derived cells were suspended in PBS containing 10% trypsin-EDTA to prevent aggregation. Flow cytometric analysis was performed on a BD Biosciences LSRII-561 system where threshold forward scatter (FSC) and side scatter (SSC) parameters were set so that a homogenous population of live cells was counted (Fig. S4.8). FlowJo software (TreeStar) was used to analyze data and determine the indicated mean fluorescent intensity (MFI). 112 4.4.6 Generation of USF1 and USF2 knockout clonal lines USF1 KO and USF2 KO Jurkat Tat mHIV-Luciferase and wildtype Jurkat E6-1 clonal cell lines were generated by CRISPR-Cas9 gene editing. 2x106 cells were co-transfected Cas9 and gRNA targeting either genomic USF1 or USF2. Transfection was performed using the Neon Transfection System (Invitrogen) as per the manufacturer’s instructions with the following settings: Voltage, 1350 V; Width, 20 ms; Pulse number, 3x. For USF1 KO, Jurkat cells were co-transfected with Cas9-BFP (pU6-CBh-Cas9-T2A-BFP: Addgene 64323) and gRNA (pSPgRNA: Addgene 47108) containing gRNA that targeted GCCAGGTAAGGGAGGGGGCC and GGAAGACGTACTTGACGTTG. For, USF2 KO, Jurkat cells were co-transfected with Cas9-mCherry (pU6-CBh-Cas9-T2A-mCherry: Addgene 64324) and gRNA-BFP (pKLV2.2-h7SKgRNA-hU6gRNA-PGKpuroBFP: Addgene 72666) containing gRNA that targeted GTCGTGGCTGCCAGGGGCAC, GGAGGAGGGCGTCGAGCTGC, and GGCGGCCGAGGCTGTCAGCG. For USF1 KO, transfected cells were isolated by live sorting (Astrios Flow Cytometer) BFP+ cells into 96-well plates containing complete RPMI-1640 while for USF2 KO, mCherry+/BFP+ cells were isolated. Clones were expanded, and knockout of USF1 and USF2 was validated by PCR genotyping and western blotting. To generate USF2 KO TZM-bl and HEK293T clonal cell lines, cells were co-transfected using polyethylenimine (PEI) with Cas9 (pU6-CBh-Cas9-T2A-BFP: Addgene 64323) and gRNA-Puro (pKLV2.2-h7SKgRNA-hU6gRNA-PGKpuroBFP: Addgene 72666) containing gRNA that targeted GGAGGAGGGCGTCGAGCTGC and GGCGGCCGAGGCTGTCAGCG. Briefly, 6.67x105 HEK293T cells were plated with 2 mL DMEM in a 6-well plate. The following day, transfection was performed with 3 μg Cas9, 1 μg gRNA-Puro, and 12 μg PEI. Transfected cells were selected with 1 μg/mL puromycin for four days at which point single cells were plated in wells of 96-well plates by limiting dilution. Clones were expanded and USF2 KO was validated by western blotting. 4.4.7 shRNA mediated knockdown Jurkat mHIV-Luciferase cells were infected with pLKO empty vector or pLKO shRNA expressing lentivirus at a M.O.I. ~10. shRNA transduced cells were cultured for up to 8 days with 3 µg/mL puromycin during which pools of puromycin selected cells were prepared for the indicated analysis. MISSION shRNA clones (Sigma) used for knockdown are as follows: USF1, TRCN0000020679 – GCTGGATACTGGACACACTAA (3’ UTR); USF2-1, TRCN0000020734 – TCCTCCACTTGGAAACGGTAT (3’ UTR); USF2-2, TRCN0000020737 – TCCAGACTGTAACGCAGACAA (CDS); TFII-I, TRCN0000019315 – CGGATGAGTGTAGATGCTGTA (CDS). 4.4.8 ChIP-qPCR ChIP-qPCR was performed as in Chapter 2. Antibodies used are as follows: Myc (10 μg) – Santa Cruz Biotechnology sc-40, USF2 (10 μg) – Abcam ab264330, TFII-I (10 μg) – BD Biosciences 113 610942, TRIM24 (10 μg) –Proteintech 14208-1-AP. E-Box primer sequences are as follows: Fwd 5’ GTGAGCCTGCATGGAATGGA, Rev 5’ CGGATGCAGCTCTCGGG. 4.4.9 RT-PCR RNA was extracted from Jurkat Tat mHIV-Luciferase cells following the indicated treatment using RNeasy Kit (Qiagen). RNA was analyzed using the Quant Studio 3 Real-Time PCR system (Applied Biosystems) using Power SYBR® Green RNA-to-CT™ 1-Step Kit (Thermo Fisher) as per the manufacturer’s instructions. RT-PCR data was normalized to GAPDH expression using the ΔΔCt method. Primers used are as follows: USF1 Fwd 5’ GCTCTATGGAGAGCACCAAGTC, Rev 5’ AGACAAGCGGTGGTTACTCTGC; GAPDH Fwd 5’ TGCACCACCAACTGCTTAGC, Rev 5’ GGCATGGACTGTGGTCATGAG. 4.4.10 RNA-Seq RNA was extracted from wildtype, USF1 KO, or USF2 KO Jurkat Tat mHIV-Luciferase human T cells that were left untreated or stimulated with PMA/ ionomycin for 4 hrs. Sample quality control was performed using an Agilent 2100 Bioanalyzer. Qualifying samples were prepped using NEBnext Ultra ii Stranded mRNA (New England Biolabs) standard protocol. Sequencing was performed on the Illumina NextSeq 500 with Paired End 42bp × 42bp reads. De-multiplexed read sequences were uploaded to the Galaxy web platform (Afgan et al, 2018) and aligned to the hg38 reference genome using STAR. Subsequently, transcript assembly was accomplished with featureCounts and differential gene expression (DEG) was determined by DESeq2 analysis. DEG are defined as having a fold change in expression > 1.5 and p-value < 0.05 as to the compared condition. Volcano plots were generated on the Galaxy web platform and gene expression heatmaps were created in RStudio. 4.4.11 Statistics and reproducibility All replicates are independent biological replicates and are presented as mean values with ± standard deviation shown by error bars. The number of times that an experiment was performed is indicated in the figure legends. P-values were determined by performing unpaired samples t-test or ANOVA, as indicated, with the use of GraphPad Prism 9.0.0. Statistical significance is indicated at *P < 0.05, **P < 0.01, or ***P < 0.001, with n.s. denoting non-significant P ≥ 0.05. 4.4.12 Data availability All data supporting the findings of this study are available within the article (Horvath et al, 2023) or from the corresponding author upon reasonable request (I. Sadowski, ijs.ubc@gmail.com). All high-throughput RNA-seq data has been deposited with NCBI GEO, accession GSE227850. 114 4.5 Chapter acknowledgments We thank Andy Johnson and Justin Wong of the UBC Flow Cytometry Facility for performing FACS as well as for assistance with flow cytometry. This research was supported by program project grant F16-01210, from the Canadian Institutes of Health Research. 4.6 Supplementary figures Figure S4.1 shRNA knockdown of USF2 suppresses HIV-1 expression. A, C: Jurkat mHIV-Luciferase cells were transduced with an pLKO empty vector control or shRNA targeting USF1 (A) or USF2 (C). Transduced cells were selected for with 3 μg/mL puromycin for up to 8 days after which lysates were analyzed by immunoblotting using antibodies targeting USF1, USF2, and Tubulin. B, D: USF1 (B) or USF2 (D) shRNA transduced mHIV-Luciferase cells were left untreated (Ve, DMSO) or stimulated with 5 nM PMA for 4 hours prior to measuring luciferase activity (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 115 Figure S4.2 USF2 knockout limits proviral induction in TZM-bl cells. A: USF2 KO in the TZM-bl cell line was produced by using CRISPR-Cas9. Immunoblotting of cellular lysates was performed using antibodies against USF2 and Tubulin. B: Wildtype or USF2 KO TZM-bl cells were left untreated (Ve, DMSO) or incubated with 5 nM PMA for 4 hours prior to measuring luciferase activity. The relative light units (RLU) are displayed (n = 3, mean ± SD). 116 Figure S4.3 Reincorporation of USF1 or USF2 rescues LTR transcription in USF2 KO cells. A: Schematic representation of the LAI LTR-Tat-IRES-GFP reporter construct as well as the CMV-EV-EF1α-RFP (EV), CMV-USF1-EF1α-RFP (USF1), and CMV-USF2-EF1α-RFP (USF2) vectors. B: Depiction of the flow cytometry scatter plot generated following co-transfection of LTR reporter and RFP expression vector. The bottom left quadrant contains un-transfected cells, negative fluorescent cells. The top left population is co-transfected by the LTR reporter and the RFP expression vector but the LTR is silent. Cells in the top right quadrant are 117 co-transfected by transcriptionally active LTR reporter as well as the RFP expression vector. The bottom right quadrant are cells transfected only by the active LTR reporter. C: Representative scatter plots following co-transfection of wildtype or USF2 KO HEK293T cells with the LTR- Tat-IRES-GFP and either CMV-EV-EF1α-RFP, CMV-USF1-EF1α-RFP (USF1), or CMV-USF2-EF1α-RFP (USF2) vector. Figure S4.4 Loss of USF1 or USF2 does not affect cellular growth. A, B: Equivalent number of Wildtype, USF1 KO (A), and USF2 KO (B) Jurkat mHIV-Luc cells were plated, and cell number counted on the indicated day (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C, D: As in A, B but cellular viability was determined (n = 3, mean ± SD). 118 Figure S4.5 USF2 regulates HIV expression regardless of Tat. A: Schematic representation of the CMV-EV-EF1α-RFP vector and the LAI LTR-IRES-GFP (-Tat) and LTR-Tat-IRES-GFP (+Tat) reporter constructs. B: Depiction of the flow cytometry scatter plot generated following co-transfection of LTR reporter and RFP expression vector. See Fig. S4.3B for further description. C, D: Representative scatter plots following co-transfection of wildtype or USF2 KO HEK293T cell lines with RFP expression vector and either LTR-IRES-GFP (C) or LTR-Tat-IRES-GFP (D) LAI derived reporter construct. 119 Figure S4.6 Loss of either USF1 or USF2 hinders expression of genes downstream of multiple T cell activation pathways. A - D: Hallmark GSEA plots of RNA-seq data analyzed by DESeq2 of PMA/ionomycin stimulated USF1 KO and USF2 KO Jurkat T cells. Analysis was performed on n = 3 RNA-seq samples. 120 Figure S4.7 USF2 mediates HIV-1 infection of T cells. A: representative scatter plots of Wildtype, USF1 KO, USF2 KO Jurkat Tat T cells 3 days post-infection with Red-Blue-HIV-1 dual reporter virus (Matthew S Dahabieh et al. 2014). LTR driven BFP expression is on the x-axis while mCherry expression driven by the constitutive CMV promoter is on the y-axis. B: Wildtype, USF1 KO, or USF2 KO Jurkat Tat cells were infected at the same multiplicity of infection (M.O.I. ~0.1). 3 days post-infection, HIV-1 was assessed by flow cytometry and the infection rate was normalized to the number of wildtype cells that were infected (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: Same as in B, but the ratio of productive to latent infection was determined (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 121 Figure S4.8 Representative flow cytometry gating strategy. A, B: Representative scatter plots depicting the gating strategy employed for HEK293T cell lines (A) and Jurkat T cell lines (B). Homogeneous populations of live cells were isolated and assessed by setting threshold forward scatter (FSC) and side scatter (SSC) settings. 122 Chapter 5: CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells 5.1 Introduction Expression of HIV-1 provirus in T cells is regulated by multiple signaling pathways that respond to cytokines, innate immune response, and engagement of the T cell receptor with antigen presenting dendritic cells. Response to these signals is controlled by sequence-specific transcriptional activator proteins bound to the 5' LTR enhancer and control regions, that include, but is not limited to NF-B, NFAT, GABP/ Ets, STAT1/3, AP1, TCF-1/ Lef, and TFII-I-USF1/2 (RBF-2) (Sadowski & Mitchell, 2005) (Sadowski et al, 2008). These factors stimulate transcription from the HIV-1 LTR promoter through interactions that recruit general TFs (GTFs), including TFIID, mediator, and RNA Polymerase II (Karn & Stoltzfus, 2012). Several factors bound to the LTR, specifically TFII-I/ USF1/2 (RBF-2) also promote elongation of transcripts from the core promoter by mechanisms involving recruitment of CDK9/ P-TEFb through interaction with the co-activator TRIM24 (Horvath et al, 2023) (Horvath et al, 2023). Multiple transcriptional activators that bind the 5' HIV-1 LTR were shown to be regulated by the mediator kinase CDK8/19, including NF-B (Chen et al, 2017), STAT1/3 (Bancerek et al, 2013), and TCF-1/ LEF- catenin (Firestein et al, 2008). Because these factors play central roles in response to signals that regulate HIV-1 expression (Acchioni et al, 2019) (Barbian et al, 2022) (Bosque et al, 2017) we have examined the effect of Cdk8 inhibitors and CDK8 gene knockouts on HIV-1 provirus expression in T cells. In this chapter, we examine the effect of CDK8/19 inhibitors and CDK8 knockouts on expression of HIV-1 provirus. We find that Senexin A and BRD6989, structurally distinct chemical inhibitors specific for CDK8/19 kinase activity, impair reactivation of HIV-1 provirus in cell line models of latency, and encourage establishment of immediate latency in newly infected T cells. Inhibition of CDK8/19 impairs recruitment of RNA Pol II to the LTR promoter. Furthermore, CRISPR-mediated knockout of the CDK8 gene impairs the expression and activation of HIV-1 in response to several latency reversal agents. Interestingly however, disruption of CDK8 does not entirely mimic the effect of CDK8/19 kinase inhibition, indicating that CDK8 and CDK19 do not function redundantly for the activation of proviral transcription. Collectively, our observations indicate that CDK8/19 inhibitors, including those currently in clinical trials for treatment of various cancers, may prove useful for therapies to eliminate latently infected cells by suppressing HIV-1 expression. 5.2 Results 5.2.1 Chemical inhibition of CDK8/19 kinase activity suppresses HIV-1 expression We examined the effect of small molecule inhibitors of CDK8/19 kinase activity on HIV-1 expression by treating the previously characterized JLat10.6 (Read et al, 2019) and mHIV-Luciferase (Bernhard et al, 2011) Jurkat human CD4+ T cell lines with two structurally unrelated CDK8/19 inhibitors, Senexin A and BRD6989 (Fig. 5.1A) (Johannessen et al, 2017) (Porter et al, 123 2012). JLat10.6 Jurkat cells possess a full-length HIV-1 provirus with GFP expressed instead of Nef from the 5' LTR (Jordan et al, 2003), while Jurkat Tat mHIV-Luciferase cells express luciferase as a fusion with gag from the 5' HIV-1 LTR, but no other HIV-1 proteins (Fig. 5.1B) (Bernhard et al, 2011) (Horvath et al, 2023). We did not observe an effect on basal GFP expression in JLat10.6 cells upon treatment with these inhibitors, but induction of GFP expression in cells treated with the PKC agonist PMA was inhibited in a concentration-dependent manner (Fig. 5.1C, 5.1D). A similar effect was seen with mHIV-Luciferase cells, although with this reporter line we also recorded a significant reduction in basal luciferase expression upon treatment with Senexin A but not with BRD6989 (Fig. 5.1E, 5.1F). The inhibitory effect of the CDK8/19 inhibitors on HIV-1 expression was not due to toxicity, as concentrations as high as 100 M had only minor effects on cell viability (Fig. S5.1A, S5.1B). Additionally, we examined effect of the CDK8/19 inhibitors on expression of HIV-1 provirus in the ACH2 CEM CD4+ T cell and U1 monocyte models of latency, where we likewise observe inhibition of basal and PMA-induced expression of viral mRNA (Fig. 5.2A, 5.2B) and minimal toxicity at concentrations where inhibition was detected (Fig. S5.1C, S5.1D). These observations indicate that chemical inhibition of CDK8/19 kinase activity suppresses HIV-1 expression in several cell line models of provirus latency. 124 Figure 5.1 CDK8/19 inhibitors impair reactivation of HIV-1 provirus. A: Structure of CDK8/19 inhibitors Senexin A and BRD6989. B: Schematic representation of integrated reporter virus in the JLat10.6 (top) and mHIV-luciferase (bottom) Jurkat cell lines. The JLat10.6 reporter represents the full HIV-1 genome with GFP inserted into nef, while mHIV-luciferase cells express luciferase as a fusion with gag from the 5' LTR, with a provirus that is deleted of all other viral encoded proteins. C, D: JLat10.6 cells were pre-treated for 1 hr with the indicated concentration of Senexin A (C) or BRD6989 (D). Subsequently, 10 nM PMA was added and flow cytometry was performed following 20 hrs of incubation (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. E, F: mHIV-Luciferase cells were pre-treated for 1 hr with the indicated concentration of Senexin A (E) or BRD6989 (F). Following pre-treatment, 10 nM PMA was added, and luciferase assays were performed after 4 hrs. Results are normalized to the maximum luciferase expression recorded, i.e., PMA induced mHIV-Luciferase cells that 125 were not treated with any amount of Senexin A or BRD6989 (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. Figure 5.2 CDK8/19 inhibitors prevent reactivation of HIV-1 in cell models of HIV-1 latency. A, B: ACH2 (A) or promonocytic U1 cells (B), were incubated with a DMSO vehicle control (Ve), 10 μM Senexin A, or 10 μM BRD6989 for 1 hr. Afterwards, DMSO (Ve) or 10 nM PMA was added, and cells were incubated for 20 hrs prior to extraction of intracellular RNA. Expression of HIV-1 mRNA was measured by performing RT-PCR with oligos that detect multiply spliced Tat-Rev transcripts and normalizing to GAPDH (n = 2 - 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 5.2.2 CDK8/19 antagonizes HIV-1 reactivation by latency reversing agents We examined the effect of CDK8/19 inhibitors on stimulation of HIV-1 expression by additional signaling agonists. Treatment with a combination of the phorbol ester PMA and ionomycin mimics T cell activation by stimulation of the MAPK, PKC and calcineurin signaling pathways downstream of the T cell receptor (Chen et al, 2005) (Malcolm et al, 2007). PMA stimulates PKC, which in T cells activates the MAPK and IKK-IκB-NFκB pathways, while ionomycin causes release of intracellular calcium, and activation of NFAT though dephosphorylation by calcineurin (Sadowski & Mitchell, 2005). We found that HIV-1 expression in both the JLat10.6 and mHIV-Luciferase cell lines treated with PMA, alone or in combination with ionomycin, is reduced by Senexin A and BRD6989 (Fig. 5.3A, 5.3B). Similarly, the CDK8/19 inhibitors also significantly impair HIV-1 activation in response to Ingenol 3-angelate (PEP005) (Fig. 5.3A, 5.3B), a latency reversal agent (LRA) which activates NF-κB (Acchioni et al, 2019) (Jiang et al, 2015). Latent HIV-1 provirus can also be reactivated by treatments which affect chromatin modifications, or association of epigenetic modifiers with the LTR in the absence of signaling 126 agonists (Hashemi & Sadowski, 2019). The histone deacetylase inhibitor (HDACi) suberanilohydroxamic acid (SAHA) for example, as well as the BRD4 inhibitor JQ1, are well characterized latency reversing agents (LRAs) (Jiang et al, 2015) (Desimio et al, 2017). SAHA induces expression of HIV-1 to a comparable level as PEP005 in the JLat10.6 cell line, and we found that this effect was also partially inhibited by Senexin A and BRD6989 (Fig. 5.3A). The effect of SAHA on HIV-1 expression in the mHIV-Luciferase line was not as pronounced, and here we observed only a minor but significant effect of the CDK8 inhibitors (Fig. 5.3B). JQ1 caused significantly lower levels of reactivation in both reporter cell lines, which was only significantly affected by the CDK8/19 inhibitors in the mHIV-Luciferase cell line (Fig. 5.3A, 5.3B). These results demonstrate that CDK8/19 kinase activity is required for the most robust reactivation of HIV-1 expression in response to a variety of LRAs. Figure 5.3 Inhibition of CDK8/19 impairs reactivation of HIV-1 by latency reversing agents. A, B: JLat10.6 (A) or mHIV-Luciferase (B) cells were treated with a DMSO vehicle 127 control (Ve), 10 μM Senexin A, or 10 μM BRD6989 for 1 hr. Next, cells were left untreated (Un) or treated with 10 nM PMA/ 1 μM ionomycin, 10 nM PMA, 10 nM PEP005, 10 μM SAHA, or 10 μM JQ1. GFP expression of JLat10.6 cells was determined by flow cytometry following 20 hrs of incubation (n = 2 - 3, mean ± SD). mHIV-Luciferase cells were treated as in (A), but LTR expression was determined following 4 hrs treatment by luciferase assay (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 5.2.3 Effect of CDK8/19 inhibition on cell growth Above, we investigated the effect of Senexin A and BRD6989 on HIV-1 expression by employing temporally acute treatments, however, HIV-1 infection is recognized as a chronic disease that may require prolonged intervention. To further examine the cellular impact of CDK8/19 inhibition, we assessed the growth of Jurkat T cells exposed to a range of drug concentrations for four days (Fig. 5.4). On day 3, 10 μM of Senexin A was found to slightly impair cell expansion while 50 μM of the compound elicited cell cycle arrest (Fig. 5.4A). In contrast, cell cycle progression was less affected by BRD6989 with concentrations as high as 50 μM having minor observable effects on cell division (Fig. 5.4B). Importantly, no effect on cellular viability throughout the four days examined was observed for any of the employed Senexin A or BRD6989 concentrations (Fig. 5.4C, 5.4D). 128 Figure 5.4 Effect of Senexin A and BRD6989 on cellular growth and viability. A, B: Jurkat T cells were incubated with the indicated concentration of Senexin A (A) or BRD6989 (B) or a vehicle control (Ve, DMSO) for four days. Cells were counted on the indicated day for the given treatment (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. B, C: Jurkat T cells treated as in A, B were assessed for viability (n = 3, mean ± SD). 5.2.4 Inhibition of CDK8/19 encourages establishment of HIV-1 latency Having determined concentrations of Senexin A and BRD6989 that have no observed toxicity and minimal impact on cell cycle progression, we next examined the role of CDK8/19 for establishment of immediate latent infection in Jurkat T cells using the replication incompetent Red-Green-HIV-1 (RGH) virus. RGH is a dual reporter virus where GFP is expressed from the 5’ LTR and mCherry from an internal constitutive PGK promoter, thus allowing latently infected (mCherry+/GFP-) cells to be discriminated from productively infected 129 (mCherry+/GFP+) cells as early as 24 hours post infection by flow cytometry (Fig. 5.5A, 5.5B) (Dahabieh et al, 2013) (Dahabieh et al, 2014). Consistent with previous observations (Dahabieh et al, 2013) (Hashemi et al, 2016), the proportion of productively infected cells peaks at 4 days post infection, and then declines as the provirus succumbs to silencing (Fig. 5.5B, Ve) (Hashemi et al, 2016). In contrast, infection of cells treated with Senexin A or BRD6989 over the course of the analysis produced a significantly lower proportion of productively infected cells, with the largest discrepancy occurring at 7 days post-infection (Fig. 5.5B, 5.5C). Similar results were observed with infection of the SupT1 human T cell line (Fig. S5.2A, S5.2B), and with an additional dual HIV-1 reporter virus (Fig. S5.2C, S5.2D) (Battivelli et al, 2018). These results indicate that inhibition of CDK8/19 kinase activity in newly infected cells encourages production of immediate latent provirus. Additionally, we examined whether treatment of cells with the CDK8/19 inhibitors post-infection affected the proportion of latent or productively infected cells. For this, we treated cells with the CDK8/19 inhibitors 4 days post-infection and measured the proportion of latently and productively infected cells 24 hours later (Fig. 5.5D). In this experiment we found that Senexin A caused a ~20% reduction in productive infections, while BRD6989 caused a more modest ~10% decrease as compared to cells treated with a vehicle control (Fig. 5.5E). This result indicates that effects of the CDK8/19 inhibitors are not temporally restricted to immediate events in HIV-1 infection, including entry and formation of integrated provirus, but rather must inhibit expression of the virus post-integration, which is consistent with the effect of these compounds on reactivation of provirus reporter by latency reversing agents. 130 Figure 5.5 Inhibition of CDK8/19 promotes establishment of latency in newly infected T cells. A: Flow cytometric analysis of cells infected with the single-round, Red-Green-HIV-1 (RGH) dual reporter virus produces a scatter plot with distinct populations (Dahabieh et al, 2013). Bottom left contains the uninfected (fluorescent negative) cells, top left is the population of latently infected cells (mCherry+/GFP-), top right displays the productive infections (mCherry+/GFP+), while bottom right contains noise generated from viral rearrangements (mCherry-/GFP+). B: Representative flow cytometry scatter plots of Jurkat T cells that are uninfected or are 7 days post RGH infection while being treated with a vehicle control (DMSO), 10 μM Senexin A, or 10 μM BRD6989. C: Untreated (Ve, DMSO) or Jurkat T cells treated with 10 μM Senexin A or 10 μM BRD6989 were infected with RGH, and the percentage of productively infected cells was determined at the indicated times post-infection by flow cytometry. Values are normalized to the ratio of productive infections of the vehicle control (Ve) (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. D: The effect of 131 treatment with CDK8/19 inhibitors post-infection was determined by the addition of 10 μM Senexin A or 10 μM BRD6989 to Jurkat cells 4 days post-infection with RGH, and the proportion of cells with productive infections was determined 20 hours later by flow cytometry. E: The proportion of productively infected cells treated 4 days post-infection with 10 μM Senexin A or 10 μM BRD6989, was determined by flow cytometry. Values are normalized to the ratio of productive infections of the vehicle control (Ve) (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 5.2.5 CDK8/19 inhibitors encourage HIV-1 latency in primary CD4+ T cells ex vivo We examined the effect of Senexin A and BRD6989 on HIV-1 replication in primary CD4+ T cells. For this, we treated CD4+ T cells from normal donors with the CDK8/19 inhibitors or vehicle control prior to single-round infection with the RGH dual-reporter virus. Proviral activity was assessed by flow cytometry 3 days post-infection (Fig. 5.6A). Consistent with the results described above with reporter cell lines and infection of Jurkat T cells, we found that Senexin A and BRD6989 cause a significant decrease in the proportion of productively infected cells, represented by expression of both mCherry and GFP as compared to mCherry alone (Fig. 5.6B), where Senexin A inhibited to a greater effect than BRD6989 (Fig. 5.6C). Furthermore, we found that proviruses that managed to establish productive infections in the presence of Senexin A or BRD6989 displayed lower levels of transcriptional activity than their untreated counterparts (Fig. 5.6D, Productive Inf.). Notably, the CDK8/19 inhibitors did not affect the viability (Fig. 5.6E) and only had a slight impact on division (Fig. 5.6F) of the primary CD4+ T cells over the course of the treatment, similar to the results obtained from Jurkat T cells (Fig. 5.4). Finally, CD4+ T cells treated with ethe CDK8/19 inhibitors showed no difference in susceptibility to HIV-1 infection (Fig. 5.6G). Collectively, these results indicate that Senexin A and BRD6989 inhibit HIV-1 expression in primary T cells, an effect which encourages the formation of latency. 132 Figure 5.6 HIV-1 latency is regulated by CDK8/19 kinase activity in primary CD4+ T cells. A: Following a 1 hr pre-treatment with a vehicle control (Ve, DMSO), 10 μM Senexin A, or 10 133 μM BRD6989, CD4+ PBMC T cells were infected with RGH. Flow cytometry was performed after three days of treatment. B: Representative flow cytometry scatter plots of primary CD4+ T cells infected with RGH. GFP expression is indicated on the x-axis and mCherry on the y-axis. Q1 is the population of cells that is latently infected (mCherry+/GFP-), Q2 displays productive infections (mCherry+/GFP+), Q3 depicts noise generated from viral rearrangements (mCherry-/GFP+), and Q4 displays the uninfected population (mCherry-/GFP-). C: RGH infected CD4+ T cells were examined by flow cytometry 3 days post-infection. Values are normalized to the ratio of productive infections of the vehicle control (Ve) (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. D: As in (C) but the Mean Fluorescence Intensity (MFI) of GFP expression is assessed. GFP MFI was evaluated within the entire population of infected cells (All Infections, mCherry+/GFP- and mCherry+/GFP+) or within the population of cells that harbored productive infections (Productive, mCherry+/GFP+) (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. E: Viability of cells treated as in (A) (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. F: CD4+ T cells treated as in (B) were counted on the indicated day post-infection (n = 3, mean ± SD). G: The infection rate was determined for primary T cells as treated in (B). Values are normalized to the vehicle control (Ve) (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. 5.2.6 Senexin A and BRD6989 do not inhibit HIV-1 Tat, and block HIV reactivation in a cell-specific manner Originally isolated from the murine sponge Corticium simplex (Aoki et al, 2006), the steroidal alkaloid cortistatin A (CA) was found to inhibit CDK8/19 kinase activity (Cee et al, 2009) (Pelish et al, 2015). Because of its limited availability from nature, analogs of CA were developed, including didehydro-Cortistatin A (dCA) (Shi et al, 2009) (Shi et al, 2011). Subsequently, dCA was found to suppress HIV-1 expression by a mechanism proposed to involve inhibition of Tat, the HIV-1 transactivator of transcription, independently from effects involving inhibition of CDK8/19 activity (Kessing et al, 2015) (Mediouni et al, 2019) (Mousseau et al, 2012) (Mousseau et al, 2015). Given these reported effects of dCA on HIV-1 Tat, we examined whether the CDK8/19 inhibitors Senexin A and BRD6989 had a similar effect on Tat function. To examine this, we transfected HEK293T cells with an HIV-1 LTR reporter plasmid where GFP is expressed from an IRES, alone or in combination with viral Tat protein (Fig. 5.7A). As expected, transfection of HEK293T cells with the reporter co-expressing Tat resulted in substantially elevated GFP expression compared to the reporter lacking Tat (Fig. 5.7B). Interestingly however, neither of the CDK8/19 kinase inhibitors had an effect on expression of GFP from either of these reporters (Fig. 5.7B), indicating that these compounds likely do not affect Tat function, unlike the reported effect of dCA (Mediouni et al, 2019) (Kessing et al, 2015). Consistent with this observation, we note that HIV-1 provirus in the ACH2 cell line possesses a point mutation in TAR rendering it defective to Tat transactivation (Emiliani et al, 1996), while the provirus in U1 cells expresses defective Tat protein (Emiliani et 134 al, 1998). As shown above (Fig. 5.2), Senexin A and BRD6989 both inhibit reactivation of HIV-1 expression in these lines. Furthermore, Senexin A and BRD6989 are not structurally related to dCA (Johannessen et al, 2018) (Porter et al, 2012), and these results indicate they must have distinct mechanisms of action. Considering that the CDK8/19 inhibitors antagonize HIV-1 expression in Jurkat T cells but did not affect basal or Tat-activated expression of the LTR-IRES-GFP reporter in HEK293T cells (Fig. 5.7B), we wondered whether the effect of these compounds on HIV-1 expression may be cell type-specific. To examine this, we used the HeLa-derived TZM-bl cell line which bears an integrated HIV-1 LTR--Gal-Luciferse reporter (Wei et al, 2002). We observed induction of luciferase expression in this line in response to PMA, or the LRAs PEP005, SAHA and JQ1. However, treatment with Senexin A or BRD6989 had no effect on this response (Fig. 5.7C). These observations indicate that effects of CDK8/19 kinase activity on HIV-1 expression may be specific to cells of leukocyte lineage. Relating to this, we note that it was previously reported that knockdown of CDK8 and/or CDK19 does not affect HIV-1 infection in HeLa cells, where dCA has an inhibitory effect (Mediouni et al, 2019). This supports a view that dCA must have additional inhibitory target(s) apart from the CDK8/19 kinase such as Tat, and that the effect of mediator kinase for HIV-1 expression may be unique to T cells. Figure 5.7 CDK8/19 inhibitors do not affect HIV-1 transactivation by Tat. A: Schematic representation of HIV-1 LAI derived LTR-IRES-GFP (-Tat) and LTR-Tat-IRES-GFP (+Tat) reporter constructs. B: HEK293T cells were transfected with the GFP reporters; one day post-transfection, cells were either left untreated (Ve) or treated with 10 μM Senexin A or 10 μM BRD6989. Flow cytometry was performed 20 hr later with the Mean Fluorescence Intensity (MFI) of the GFP+ population being measured (n = 2, mean ± SD). C: TZM-bl Hela-derived 135 cells possessing an integrated LTR-luciferase reporter were pre-treated for 1 hr with DMSO vehicle control (Un), 10 μM Senexin A, or 10 μM BRD6989. The pre-treated cells were then stimulated with 10 nM PMA, 10 nM PEP005, 10 μM SAHA, or 10 μM JQ1. Luciferase expression was measured following 4 hrs of treatment and is displayed as Relative Light Units (RLU) (n = 3, mean ± SD). 5.2.7 Inhibition of CDK8/19 inhibits recruitment of RNA PolII, but promotes association of the mediator kinases with the LTR Regulation of transcription from the HIV-1 LTR, like most cellular genes, involves the recruitment of RNA Pol II and histone modifying complexes to the promoter as well as regulation of transcriptional elongation and mRNA processing (Judge et al, 2020) (Sadowski & Hashemi, 2019). To examine which of these steps in HIV-1 expression may be affected by CDK8/19 kinase inhibition, we assessed recruitment of RNA PolII to the LTR using ChIP-qPCR. We found that treatment of unstimulated cells with Senexin A caused a significant decrease in association of RNA Pol II at the LTR RBE3 and RBE1 cis-elements located upstream of the enhancer region and near the transcriptional start site, respectively (Fig. 5.8A, compare Ve and SenA). Treatment with PEP005, a PKC agonist which imparts activation of NF-κB, resulted in a significant increase in LTR associated RNAPII (Fig 5.8A, compare Ve and PEP005), but this effect is blocked by the CDK8/19 inhibitor Senexin A (Fig. 5.8A, compare PEP005 and PEP005/SenA). These results indicate that CDK8/19 kinase activity is necessary for efficient recruitment of RNA Pol II to the LTR. Next, we examined the effect of CDK8/19 kinase inhibition on localization of CDK8 and CDK19 to the HIV-1 LTR. Treatment of cells expressing Flag-tagged CDK8 or CDK19 with Senexin A does not appear to affect abundance of these proteins (Fig. 5.8B), but interestingly, we observe increased association of both kinases with the LTR in cells treated with this inhibitor (Fig. 5.8C, 5.8D). It is possible that direct interaction of the CDK8/19 Kinase Module (CKM) negatively regulates HIV expression, which is consistent with a previous report indicating that CDK8 is evicted from the HIV-1 LTR upon T cell stimulation and induction of virus expression (Kim et al, 2006). These observations would support a view that interaction of mediator kinase module with the promoter has a negative regulatory effect on transcription, a function that was also indicated from experiments with yeast (Jeronimo et al, 2016) and transcription reactions in vitro (Osman et al, 2021). However, decreasing promoter transcriptional activity through TFIIH inhibition was found to enrich core promoter associated Mediator in yeast (Jeronimo et al, 2016). It is possible that suppressed LTR transcription in response to CDK8/19 inhibition is altering the kinetics of these proteins and allowing our ChIP experiment to capture excess Flag tagged CDK8 or CDK19. 136 Figure 5.8 Inhibition of CDK8/19 impairs recruitment of RNA Polymerase II to the HIV-1 LTR. A: JLat10.6 cells were incubated for 20 hrs with a DMSO vehicle control (Ve), 10 μM Senexin A, 10 nM PEP005, or were pre-treated for 1 hr with 10 μM Senexin A, after which 10 nM PEP005 was added. ChIP was performed using an anti-RNAPII antibody and analyzed with qPCR with primers specific for the HIV-1 LTR RBE1 and RBE3 regions, or an intragenic Gag site. ChIP-qPCR results are normalized by subtraction of values produced with sample paired non-specific IgG immunoprecipitation (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. B: JLat10.6 cells were transduced with a non-expressing lentivirus (EV, lanes 1 and 2) or lentiviral vectors expressing Flag-tagged CDK8 (lanes 3 and 4) or Flag-tagged CDK19 (lanes 5 and 6). Transduced cells were left untreated (Sen A -) or incubated for 20 hrs with 10 μM Senexin A (Sen A +) after which cellular lysate was extracted and immunoblotted with antibodies against Flag and Tubulin. C, D: JLat10.6 cells expressing Flag-tagged CDK8 (C) or Flag-tagged CDK19 (D) were treated with DMSO vehicle control (Ve) or 10 μM Senexin A. Following 20 hrs incubation, ChIP was performed using anti-Flag antibody, and qPCR was used to detect enrichment at the RBE3 and RBE1 LTR sites. ChIP-qPCR results are normalized by subtraction of values produced with sample paired non-specific IgG immunoprecipitation (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 137 5.2.8 CDK8 and CDK19 are activators of HIV-1 expression The results above demonstrate that chemical inhibition of the CDK8/19 kinases inhibits HIV-1 basal and activated expression in Jurkat T cells and normal CD4+ lymphocytes. All current small molecule inhibitors of the mediator kinases do not differentiate between effects on CDK8 and CDK19 (Wu et al, 2021) (Yu et al, 2021). Although these kinases are generally considered to have overlapping or redundant function for the mediator kinase module (Fant & Taatjes, 2019) (Wu et al, 2021), the role of CDK19 is less well understood and some evidence suggests specific functions for CDK19 (Zhang et al, 2022). To assess the specific role of CDK8 in regulating HIV-1 expression, we used shRNA mediated knockdown of CDK8 in both the mHIV-Luciferase (Fig. S5.3A) and JLat10.6 (Fig. S5.3C) cell lines. We found that knockdown of CDK8 by shRNA in the mHIV-Luciferase cell rendered the integrated provirus significantly impaired for PMA induced luciferase reporter expression (Fig. S5.3B). Curiously however, we found that depletion of CDK8 expression by shRNA in the JLat10.6 cell line did not inhibit induction of GFP upon PMA treatment (Fig. S5.3D). To examine the effect of CDK8 on HIV-1 transcription in more detail, we performed CRISPR-Cas9 gene editing to generate mHIV-Luciferase (Fig. 5.9A, lanes 2-7) and JLat10.6 (Fig. 5.9A, lanes 9-11) CDK8 knockout (CDK8 KO) cell lines. Consistent with results from shRNA knockdown, we found that all of the CDK8 KO mHIV-Luciferase cell lines displayed a significant defect for reactivation of expression in response to PMA (Fig. 5.9B). However, likewise to results with shRNA knock down, we did not observe an effect of the CDK8 KO in JLat10.6 cells on proviral expression in response to PMA (Fig. 5.9C). These observations indicate that CDK8 function is required for reporter gene expression in the mHIV-Luciferase but not in the JLat10.6 line. Considering that the chemical inhibitors of CDK8/19 inhibit PMA induced HIV-1 expression in both of these lines (Fig. 5.1), the differential effect of CDK8 disruption in the mHIV-Luciferase and JLat10.6 lines may indicate functional differences between CDK8 and CDK19, a possibility we discuss further below. We do note that JLat10.6 cells typically express slightly higher levels of CDK8 protein than mHIV-Luciferase cells as determined by immunoblotting (Fig. 5.9A, Fig. S5.4C). 138 Figure 5.9 Effect of CDK8 gene knockout on PMA induced HIV-1 expression. A: CDK8 knockout clones were generated in the mHIV-Luciferase (lanes 2-7) and JLat10.6 (lanes 9-11) cell lines. Cell lysates from WT mHIV-luciferase (lane 1), WT JLat10.6 (lane 8), or the indicated KO clonal line were immunoblotted with antibodies against CDK8 and CDK19. (B) indicates a background signal that is produced by Rabbit antibodies against CDK19 that is unaffected by any condition we have examined. B: WT mHIV-luciferase or the CDK8 KO clones (A) were left untreated (Ve, DMSO) or stimulated with 10 nM PMA for 4 hrs after which luciferase activity was measured (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: WT JLat10.6 cells or CDK KO clones (A) were left untreated (Un) or stimulated with 10 nM PMA for 20 hours after which the proportion of GFP expressing cells was measured by flow cytometry (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. Above, we observed differential expression of HIV-1 in response to PMA between the mHIV-Luciferase and JLat10.6 Jurkat cell lines. These are two distinct clonal lines and as such, we sought to examine the effect of CDK8 abrogation on expression of a heterogenous population of provirus. For this analysis we used the replication incompetent RBH dual HIV-1 reporter which expresses all viral gene products apart from Nef and Env, and where BFP is expressed 139 from the 5' LTR, and mCherry from an internal CMV promoter (Fig. 5.10A) (Dahabieh et al, 2014). Wildtype or CDK8 KO Jurkat T cells were infected with RBH, and the populations were subsequently treated with vehicle control (Ve) or an LRA 4 days post infection; flow cytometry was performed 1 day later to examine BFP and mCherry expression (Fig. 5.10B, Fig. S5.5). Importantly, we found that the proportion of productively infected cells in the otherwise untreated population was significantly lower in CDK8 KO Jurkat cells relative to WT (Fig. 5.10C, Ve). The proportion of productively infected cells treated with the PKC agonists PMA and PEP005 was significantly increased, but interestingly, this proportion was nearly identical in the CDK8 KO line (Fig. 5.10C, PMA, PEP005). In contrast we found that treatment of RBH infected CDK8 KO cells with the histone deacetylase inhibitor SAHA, or the bromodomain inhibitors JQ1 and IACS-9571 failed to recapitulate the level of productive infections observed for wildtype cells (Fig. 5.10C, SAHA, JQ1, IACS-9571). Beyond assessing the ratio of productive versus latent infections, we took a closer examination of the effect of CDK8 KO on LTR driven BFP expression. This analysis was performed by separately evaluating the BFP mean fluorescence intensity of all infected cells (mCherry+/BFP- and mCherry+/BFP+, All Inf.) and the productive proviral infections (mCherry+/BFP+, Productive). Comparison of BFP expression of all infections showed that CDK8 KO cells were associated with decreased LTR activity upon treatment with a vehicle control (Ve), SAHA, JQ1, or IACS-9571 but no difference as compared to wildtype cells upon stimulation with PMA or PEP005 (Fig. 5.10D, All Inf.). This is expected as we are examining BFP expression for the whole of integrated proviruses and the results mirror the effect of CDK8 KO on the productive infection ratio for the indicated treatment (Fig. 5.10C). Next, in isolation, we examined LTR driven BFP expression of the proviruses that established productive infections (only the mCherry+/BFP+ population). Here, we observed a slight but non-significant decrease in viral transcription of CDK8 KO cells following treatment with PMA, PEP005, or SAHA (Fig. 5.10D). However, the productive proviral infections of CDK8 KO cells following incubation with a vehicle control, JQ1, or IACS-9571 displayed greatly reduced LTR expression as compared to wildtype (Fig. 5.10D, Productive). Collectively, these results display that CDK8 regulates basal HIV-1 transcription as well as activation in response SAHA, JQ1, and IACS-9571. However, CDK8 is not required for viral induction in response to PMA or PEP005. As the CDK8/19 dual inhibitors reduce HIV-1 expression in response to these agonists (Fig. 5.1, Fig. 5.3), CDK19 must be sufficient to mediate LTR induction in response to PKC activation. The RBH reporter virus allowed us to assess a heterogenous population of proviruses and identify latency reversal pathways that are strongly or weakly dependent on CDK8. For instance, CDK8 was not required for complete viral activation as induced by the PKC agonists PMA or PEP005 but was necessary for LTR responsiveness to the histone deacetylase inhibitor SAHA, the BRD4 bromodomain inhibitor JQ1, and the TRIM24 bromodomain inhibitor IACS-9571 (Fig. 5.10C, 5.10D). To characterize this further, we examined the effect of SAHA treatment on WT and CDK8 KO JLat10.6 cells. Although JLat10.6 cells did not require CDK8 for PMA 140 mediated reactivation (Fig. 5.9C), we found that CDK8 null cells displayed substantially dampened responsiveness of the GFP reporter to SAHA (Fig. 5.10E, 5.10F). Altogether, these results suggest that CDK8 and CDK19 may have overlapping functions for viral response to PMA or PEP005 but divergent function for reactivation by the HDAC inhibitor SAHA. 141 Figure 5.10 CDK8 knockout desensitizes HIV-1 to several latency reversal agents. A: Schematic description of the Red-Blue-HIV-1 (RBH) dual reporter assay. RBH infection produces a distinct scatter plot upon flow cytometric analysis that is similar to RGH (Fig. 5.5A), but with BFP expression being driven by the 5’ LTR instead of GFP (Dahabieh et al, 2014). We first infected wildtype or CDK8 KO Jurkat T cells with RBH. 4 days post-infection, we treated the cells with a latency reversal agent and assessed HIV-1 expression following 20 hrs of incubation. B: Representative flow cytometric scatter plots of wildtype (left) or CDK8 KO (right) Jurkat cells following incubation with DMSO (Ve) (top) or SAHA (bottom). C: Wildtype or CDK8 KO cells were treated with DMSO vehicle control (Ve), 10 nM PMA, 10 nM PEP005, 10 μM SAHA, 10 μM JQ1, or 10 μM IACS-9571, and flow cytometry was performed 20 hrs later to determine the percentage of productive infections (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. D: As in (C), but the Mean Fluorescence Intensity (MFI) of LTR driven BFP expression is depicted. All Infections measures the expression of BFP in the populations of cells that are mCherry+/BFP+ and mCherry+/BFP+ while Productive measures BFP expression in population that is mCherry+/BFP+ (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. E: WT or CDK8 KO JLat10.6 cells (A) were left untreated or incubated with 10 μM SAHA; 20 hours later flow cytometry was performed to determine the proportion of GFP+ cells (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. H: As in (G), but viral expression is reported as the Mean Fluorescence Intensity (MFI) of GFP expression (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 5.2.9 CDK8/19 inhibitors suppress HIV-1 reactivation in CD4+ T cells Because inhibition of CDK8/19 impairs HIV-1 expression in multiple models of latency and normal CD4+ T cells, we wondered if these inhibitors could also block reactivation of virus in primary CD4+ T cells from individuals with HIV-1 receiving suppressive antiretroviral therapy (ART). Treatment of CD4+ T cells from five participants with a combination PMA and ionomycin induced HIV-1 mRNA expression in all cases (Fig. 5.11A, PMA/Ion). However, Senexin A inhibition of CDK8/19 kinase activity robustly limited induction of HIV-1 viral expression by PMA and ionomycin in CD4+ T cells from all ART-treated participants (Fig. 5.11A, PMA/Ion/SenA). We observe a similar effect on expression of IL2 in these samples (Fig. 11B), which is consistent with IL2 and HIV-1 both being regulated by T cell signaling (Sadowski et al, 2008). Senexin A caused minor alterations, but more divergent effects on expression of CD69, a marker of T cell activation in these treatments (Fig. 5.11C). Importantly, Senexin A treatment had no discernable effect on cell viability at concentrations where we observe inhibition of HIV-1 provirus reactivation (Fig. 5.11D). Collectively, these observations indicate that chemical inhibitors of CDK8/19, including Senexin A, may represent potential therapies to suppress HIV-1 provirus for elimination of latently infected cells in individuals on ART. 142 Figure 5.11 Senexin A inhibits HIV-1 provirus reactivation in PBMCs from patients on ART. A - C: CD4+ PBMCs, isolated from HIV-1 infected individuals on ART, were treated for 20 hrs with DMSO (Ve), 10 μM Senexin A (Sen A), 10nM PMA and 1μM Ionomycin (PMA/ Ion), or were pre-treated with 10 μM Senexin A for 1 hr prior to the addition of 10 nM PMA/ 1μM Ionomycin. Intracellular RNA was extracted and analyzed using RT-PCR with oligos specific for multiply spliced Tat-Rev HIV-1 mRNA transcripts (A), IL2 (B), and CD69 (C). Expression of the indicated mRNA is normalized to GAPDH (per indicated patient ID, the mean of n = 2 – 3 is shown). Statistical analysis was performed using two-way ANOVA. D: Viability of cells following treatment as in (A – C) was determined (per indicated patient ID, the mean of n = 2 is shown). 143 5.3 Discussion Expression of chromosomally integrated HIV-1 provirus in T cells is dependent upon cellular factors that bind the LTR enhancer region and are regulated by signaling pathways stimulated by T cell receptor engagement (Abbas & Herbein, 2013) (Sadowski & Mitchell, 2005). Of central importance to this response is NF-B, which is activated by phosphorylation and inhibition of its cytoplasmic inhibitor I-B, allowing translocation of NF-B p65 RelA to the nucleus where it binds two sites within the LTR enhancer region and activates transcription (Pazin et al, 1996) (Williams et al, 2006). Previous work showed that CDK8/19 is co-recruited with NF-B to promoters, and that CDK8/19 inhibitors impair transcriptional activation by NF-B (Chen et al, 2017). Additional transcriptional activators regulated by CDK8 also bind the 5' HIV-1 LTR including, -catenin TCF/LEF (Firestein et al, 2008) and STAT1/3 (Bancerek et al, 2013), which is consistent with our findings that CDK8/19 inhibitors, CDK8 knockdowns, and CDK8 gene knockouts inhibit reactivation of HIV-1. We find that CDK8/19 inhibitors impair induction of HIV-1 provirus in cell line models and newly infected CD4+ T cells, and in response to a variety of latency reversing agents, including PEP005, SAHA and JQ1. Importantly, we find that the CDK8/19 inhibitor Senexin A prevents reactivation of latent virus expression in CD4+ T cells from individuals with HIV receiving suppressive antiretroviral therapy. These results indicate that chemical inhibitors of CDK8/19, some of which are presently in clinical trials for various cancers (Chen et al, 2019), could be useful to provide longstanding and durable suppression of the latent HIV-1 reservoir. We find that Senexin A impairs recruitment of RNA Polymerase II to the LTR promoter, which suggests that it causes a defect in transcriptional activation by LTR-bound transcription factors. Treatment of T cells with the PKC agonist PEP005 causes significantly enhanced association of RNA Pol II with the LTR, which likely represents the effect of LTR-associated NF-B p65 and recruitment of general transcription factor complexes, including mediator and P-TEFb (Nowak et al, 2008) (Wienerroither et al, 2015). Stimulation of NF-B p65 by TNF- was shown to cause recruitment of TFII-H/ CDK7, which was identified as a rate limiting event for reactivation of provirus from latency (Kim et al, 2006). We find that Senexin A inhibits association of RNA Pol II to the LTR in untreated cells and those stimulated with the PKC agonist PEP005 (Fig. 5.8A). Latent HIV-1 provirus is associated with RNA Pol II that is paused immediately downstream of the transcriptional start site, following synthesis of the nascent TAR RNA (Ott et al, 2011), and activation of HIV-1 involves stimulation of elongation from the viral promoter through recruitment of P-TEFb containing CDK9 and cyclin T1 (Budhiraja et al, 2013). However, the factors and mechanisms involved in establishing paused RNA Polymerase and associated GTFs at the latent HIV-1 core promoter have not been determined. Our results indicate that CDK8 activity might be required for establishment of paused RNA Pol II at the promoter. Consistent with this possibility the mediator kinase module, including CDK8 was found associated with the latent virus promoter (Kim et al, 2006). 144 The CDK8/19 inhibitors Senexin A and BRD6989 have similar effects on CDK8 and CDK19 in vitro, and presumably do not discriminate for inhibition of these paralogs in vivo (Johannessen et al, 2018) (Zhang et al, 2022). Consequently, it was interesting that the effects of CDK8 KO and CDK8/CDK19 inhibition on HIV-1 expression displayed incomplete overlap. In response to SAHA, JQ1, and IACS-9571, CDK8 ablation mimicked the effect of CDK8/19 inhibition, causing suppressed LTR transcriptional activity. However, unlike CDK8/19 inhibition, loss of CDK8 had no effect on proviral induction in response to the PKC agonists PMA or PEP005. Furthermore, activation of HIV-1 expression in CDK8 null JLat10.6 cell lines shared a similar pattern to RBH proviruses. Given these observations, it is likely that HIV-1 reactivation in response to SAHA, JQ1, and IACS-9571 is largely dependent upon CDK8, while CDK19 is generally sufficient for PKC mediated transcriptional induction. The mechanism(s) contributing to these differences are not understood, but likely represent distinct functions for CDK8 and CDK19, as Senexin A and BRD6989 inhibit HIV-1 expression in response to all of these stimuli. The mediator kinases were previously shown to have distinct functions, where it was shown that CDK19 but not CDK8 has a significant role for regulation of cell cycle progression of mouse hematopoietic stem cells (Zhang et al, 2022). We also observed that Senexin A and BRD6989 did not have an effect on HIV-1 expression in HEK293T or HeLa cells, indicating possible cell-type-specific functions for the mediator kinase. This latter result could account for a previous report indicating that the mediator kinases do not affect HIV-1 expression (Cary et al, 2019). Overall, a more detailed understanding of the mechanistic role of CDK8 and CDK19 for regulation of transcription will be required to resolve these discrepancies. Latent HIV-1 proviruses are known to produce sporadic transcripts through transcriptional noise (Singh et al, 2010) (Weinberger & Shenk, 2007), such that even the most strongly repressed provirus will occasionally produce viral transcripts (Hansen et al, 2019) (Hashemi et al, 2016). Considering these observations, it is possible that stochastic expression of latent provirus may contribute to maintenance of the latent population in tissue compartments where ART may not be capable of preventing local spread of virus (Hansen et al, 2019). Consequently, one proposed therapeutic strategy, designated block and lock, would involve intervention to suppress stochastic expression of provirus in patients on ART to promote deep latency, which may enable elimination of the latently infected population by cell lifespan decay (Pasquereau & Herbein, 2022) (Vansant et al, 2020). The results presented here indicate that inhibitors of the CDK8/19 mediator kinases may be an important contribution towards this strategy. 5.4 Materials and methods 5.4.4 Cell culture, virus culture, and lentiviral transduction Jurkat E6-1, SupT1, Jurkat Tat mHIV-Luciferase, JLat10.6, ACH2, and U1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 145 penicillin [100 units/ml], streptomycin [100 g/ml], and L-glutamine [2 mM]. HEK293T and TZM-bl cells were cultured in Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% fetal bovine serum (FBS), penicillin [100 units/ml], streptomycin [100 g/ml], and L-glutamine [2 mM]). All cell lines were incubated in a humidified 37°C and 5% CO2 atmosphere. Human Peripheral Blood CD4+ T cells, purchased from STEMCELL Technologies (Catalog # 200-0165), were cultured in RPMI supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), and 30 U/mL IL2. RGH infection was performed as previously described. Briefly, cells were incubated in the presence of DynabeadsTM Human T-Activator CD3/CD28 beads for three days. Subsequently, the beads were removed, and cells infected with RGH at a multiplicity of infection (M.O.I.) causing less than 5% of the population to be infected. Peripheral Blood Mononuclear Cells (PBMC) from participants with HIV-1 on ART were isolated from whole blood by density gradient centrifugation using LymphoprepTM and SepMateTM tubes (StemCell Technologies), and cryopreserved. Upon thawing, PBMCs were cultured in RPMI supplemented with 10% FBS, penicillin [100 units/ml], streptomycin [100 g/ml], and L-glutamine [2 mM]. Samples from participants were collected with written informed consent under a protocol jointly approved by the research ethics boards at Providence Health Care/UBC and Simon Fraser University (certificate H16-02474). Vesicular stomatitis virus G (VSV-G) pseudotyped viral stocks were produced by co-transfecting HEK293T cells with a combination of viral molecular clone, psPAX, and VSV-G at a ratio of 8 μg : 4 μg : 2 μg. Transfections were performed with polyethylenimine (PEI) at a ratio of 6:1 (PEI:DNA) in Gibco Opti-MEMtm. Lentiviral infections were performed by plating 1x106 cells in 24-well plates with media containing 8 μg/mL polybrene and the amount of viral supernatant to give the desired multiplicity of infection (M.O.I.) as indicated. Plates were subsequently spinoculated for 1.5 hrs at 1500 rpm. 5.4.5 shRNA knockdown JLat10.6 or Jurkat mHIV-Luciferase cells were transduced with a pLKO empty vector or pLKO shRNA expressing lentivirus at a M.O.I. ~10. Infected JLat10.6 cells were selected by culturing in 1 μg/mL puromycin, while mHIV-Luciferase cells had media supplemented with 3 μg/mL puromycin. All experiments were performed 3 – 10 days following shRNA transduction. MISSION shRNA clones (Sigma) used for knockdown are as follows: CDK8-1, TRCN0000000489 – ATGTCCAGTAGCCAAGTTCCA (3’ UTR); CDK8-2, TRCN0000350308 – CTAACGTCAGAACCAATATTT (CDS); CDK8-3, TRCN0000350344 – GCTTACCATGGACCCAATAAA (CDS). 5.4.6 CDK8 knockout CRISPR-Cas9 was used to generate clonal CDK8 knockout cells from mHIV-Luciferase and JLat10.6 cells. Cas9 (pU6_CBh-Cas9-T2A-BFP: Addgene #64323) and gRNA cassette 146 (pSPgRNA: Addgene #47108) plasmids had sequences that target genomic CDK8. These plasmids were than co-transfected into 2x106 cells using the Neon Transfection System (Invitrogen) as per the manufacturer’s instructions with the following settings: Voltage, 1350 V; Width, 20 ms; Pulse number, 3x. Knockout cells were isolated by live sorting (Astrios Flow Cytometer) BFP positive cells into 96-well plates containing complete RPMI 1640. Clones were expanded, and CDK8 KO was validated by PCR genotyping and western blotting. CDK8 gRNA target sequences were GGCCTCAGAGGCTGTGACAA and GTCTGATGTGAGTACTGTGG. 5.4.7 Immunoblotting Western blotting was performed as previously described (Hashemi et al, 2018) and full-length film is in Fig. S5.7. Antibodies were as follows: Tubulin - Abcam ab7291 (1:20000 – 1:40000), CDK8 - Abcam ab229192 (1:4000 – 1:10000), CDK19 – Sigma-Aldrich SAB4301196 (1:750), Goat Anti-Rabbit-HRP – Abcam ab6721 (1:2000000), Goat Anti-Mouse-HRP – Pierce 1858413 (1:20000). 5.4.8 Chromatin immunoprecipitation ChIP-qPCR was performed as described in Chapter 2. The following antibodies were used for IP: RNAPII - Abcam ab26721 (5 μg), Flag - Sigma Aldrich F3165 (5 μg). 5.4.9 Luciferase reporter assays For TZM-bl cells, 2x104 cells were plated with 100 μL DMEM per well in 96-well plates. Following 24 hrs, cells were incubated with the indicated concentration of drug for 4 hrs and luciferase expression was measured. For Jurkat mHIV-Luciferase cells, 1x105 luciferase expressing cells were plated with 100 µL media in 96-well plates. Luciferase activity was measured after the indicated time of treatment. Measurements were performed using the SuperlightTM luciferase reporter Gene Assay Kit (BioAssay Systems) as per the manufacturer’s instructions and 96-well plates were read in a VictorTM X3 Multilabel Plate Reader. 5.4.10 RT-PCR RNA was extracted from cells using the RNeasy Kit (Qiagen) and analyzed with the Quant Studio 3 Real-Time PCR system (Applied Biosystems) using Power SYBR® Green RNA-to-CT™ 1-Step Kit (Thermo Fisher) as per the manufacturer’s instructions. RT-PCR data was normalized to GAPDH expression using the ΔΔCt method as previously described (Livak & Schmittgen, 2001). Cycling parameters were as follows: 48 oC, 30 min, 1x; 95 oC, 10 min, 1x; 95 oC, 15 sec, 60 oC, 1 min, 60x. Primers were as follows: IL2, Fwd 5’ AACTCACCAGGATGCTCACA, Rev 5’ GCACTTCCTCCAGAGGTTTGA; CD69, Fwd 5’ TCTTTGCATCCGGAGAGTGGA, Rev 5’ ATTACAGCACACAGGACAGGA; HIV-1 mRNA (multiply spliced Tat-Rev transcripts), Fwd 5’ CTTAGGCATCTCCTATGGCAGGA, Rev 5’ GGATCTGTCTCTGTCTCTCTCTCCACC; GAPDH, Fwd 5’ TGCACCACCAACTGCTTAGC, Rev 5’ GGCATGGACTGTGGTCATGAG. 147 5.4.11 Flow cytometry Cells were treated as indicated in the figure legends. For flow cytometric analysis, human T cell lineages were suspended in PBS while HEK293T cells were suspended in PBS containing 10% trypsin-EDTA to prevent aggregation. A BD Biosciences LSRII-561 system was used for flow cytometry with threshold forward scatter (FSC) and side scatter (SSC) parameters being set so that a homogenous population of live cells was counted (Fig. S6). FlowJo software (TreeStar) was used to analyze data and determine the indicated Mean Fluorescence Intensity (MFI). 5.4.12 Statistics and reproducibility All replicates are independent biological replicates and are presented as mean values with ± standard deviation shown by error bars. The number of times that an experiment was performed is indicated in the figure legends. P-values were determined by performing unpaired samples t-test or ANOVA, as indicated, with the use of GraphPad Prism 9.0.0. Statistical significance is indicated at *P < 0.05, **P < 0.01, or ***P < 0.001, with n.s. denoting non-significant P ≥ 0.05. 5.4.13 Data availability All data supporting the findings of this study are available within the article or from the corresponding author upon reasonable request (I. Sadowski, ijs.ubc@gmail.com). RNA-seq data was obtained from NCBI GEO, accession GSE221851 (Horvath et al, 2023). 5.5 Chapter Acknowledgements We thank Andy Johnson and Justin Wong of the UBC Flow Cytometry Facility for performing FACS analysis as well as for assistance with flow cytometry. We thank the laboratory staff at the BC Centre for Excellence in HIV/AIDS for processing PBMCs from study participants. This research was supported by program project grant F16-01210, from the Canadian Institutes of Health Research (CIHR), and F19-05392 Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) (to I.S.). PBMC collection from participants with HIV-1 was supported by CIHR project grant PJT-159625 (to ZLB). ZLB is supported by a Michael Smith Health Research BC Scholar Award. Maria Aristizabal and colleagues provided valuable comments on the manuscript. 148 5.6 Supplementary figures Figure S5.1 Senexin A and BRD6989 do not affect cell viability at effective concentrations. A, B: Cell viability of JLat10.6 (A) and mHIV-luciferase (B) were determined following incubation with the indicated concentration of Senexin A or BRD6989 for 20 hrs (n = 2, mean ± SD). C, D: ACH2 (C) and U1 (D) cells were treated for 20 hrs with a DMSO vehicle control (Ve), 10 μM Senexin A, 10 μM BRD6989, 10 nM PMA, or pre-treated for 1 hr with 10 μM Senexin A or10 μM BRD6989 after which 10 nM PMA was added and incubated in combination for 20 hrs (n = 2, mean ± SD). 149 Figure S5.2 Senexin A and BRD6989 inhibit productive infection in SupT1 cells. A: SupT1 cells were left untreated (Ve) or treated with 10 μM Senexin A or 10 μM BRD6989 and infected with RGH reporter virus. The proportion of productively infected cells was determined at each indicated time point by flow cytometry. Values are normalized to the ratio of productive infections of the vehicle control (Ve) (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. B: As in (A), but cellular viability was determined at the indicated time point (n = 2, mean ± SD). C: Schematic representation of the HIV-GKO dual reporter virus where eGFP is expressed by the 5’ LTR and an internal EF1α promoter drives mKO2 expression (Battivelli et al, 2018). D: As in (A), but Jurkat cells were infected with HIV-GKO (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 150 Figure S5.3 Knockdown of CDK8 expression by shRNA inhibits reactivation of HIV-1 provirus. A: mHIV-Luciferase Jurkat cells were transduced with vector control (lane 1), or LKO vector expressing CDK8 specific shRNA (lanes 2-4). Cell lysates were immunoblotted with antibodies against CDK8 (top), or tubulin (bottom) following puromycin selection. B: mHIV-luciferase Jurkat cells infected with shRNA as in (A) were left untreated (Ve, DMSO) or treated with 10 nM PMA for 4 hrs when luciferase activity was measured (n = 3, mean ± SD). Statistical analysis was performed using unpaired t-test. C: JLat10.6 cells were transduced with vector control (lane 1), or LKO vector expressing CDK8 specific shRNAs (lanes 2-4). Cell lysates were immunoblotted with antibodies against CDK8 (top), or tubulin (bottom) following puromycin selection. D: JLat10.6 transduced with shRNA as in (C) were left untreated (Ve) or treated with 10 nM PMA for 20 hrs. Following incubation, GFP expression was analyzed by flow cytometry (n = 2, mean ± SD). Statistical analysis was performed using unpaired t-test. 151 Figure S5.4 Effect of T cell activation on CDK8/19 expression. A, B: Normalized counts of CDK8 (A) and CDK19 mRNA (B) were produced by analysis of a previous RNA-seq experiment that is deposited on NCBI GEO with the accession GSE221851 (Horvath et al, 2023). C: Jurkat mHIV-Luciferase or JLat10.6 cells were left untreated or incubated with 10 nM PMA/ 1 μM Ionomycin for 20 hrs. Cell lysate was extracted and immunoblotted with antibodies against CDK8 (top), CDK19 (middle), and tubulin (bottom). 152 153 Figure S5.5 CDK8 KO and CDK8/19 kinase inhibition differentially impacts HIV-1 expression. Scatter plots obtained following flow cytometry of RBH infected wildtype or CDK8 KO Jurkat cells as treated in Fig. 9A-D. Figure S5.6 Example of gating strategy used for flow cytometry. Threshold forward scatter (FSC) and side scatter (SSC) settings were set so that a homogenous population of living cells was assessed. 154 155 Figure S5.7 Full length western blots. 156 Chapter 6: Conclusion 6.1 TRIM24 facilitates HIV-1 transcriptional elongation Binding of USF1/2 and TFII-I at the conserved RBE3 and RBE1 sites flanking the HIV-1 LTR enhancer is essential for reactivation from latency in T cells. Here, we confirmed that TFII-I mediates proviral transcription as its knockdown renders HIV-1 provirus insensitive to T cell signaling. Using a modified yeast-two-hybrid screen (Hirst et al, 2001), we identified an interaction of TFII-I with the tripartite motif protein TRIM24 and found that TFII-I is required for TRIM24 LTR association. Similar to the effect of TFII-I depletion, loss of TRIM24 impaired reactivation of HIV-1 in response to T cell signaling. TRIM24 deficiency did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9. Additionally, A number of genomic loci are co-occupied by TRIM24/TFII-I and we found that TRIM24 deletion caused altered T cell immune response, an effect that is in part facilitated by TFII-I. Much is left to be examined regarding the role of TRIM24 for HIV-1 activation. Namely, it remains unknown how TRIM24 mediates CDK9 recruitment to the 5’ LTR (Fig. 2.10). The simplest explanation would be that TRIM24 directly interacts with P-TEFb, yet no evidence of this type currently exists. It may be possible to display direct interaction by co-immunoprecipitation. However, transcription factors and co-factors tend to be finicky or outright unamendable to this technique. Proximity labeling could be used as an alternative approach, though the assay cannot determine direct interactions and barring factor overexpression, is dependent on a degree of genome-wide association. Furthermore, TRIM24 may mediate CDK9 recruitment indirectly. Yu et al, 2019 has displayed that TRIM24 mediated Lys63-linkage ubiquitylation of CBP causes acetylation of STAT6 resulting in altered activity of the transcription factor to mediate M2 macrophage polarization. As such, it could be postulated that TRIM24 affects the activity of associated transcription factors or co-factors that in turn recruit CDK9. An exploration of the functionality of the unique TRIM24 domains would be required to begin to gain insight into this process. Another curiosity that has emerged from this work is the role of TRIM24 for enabling transcriptional elongation. Promoter proximal pausing is a widespread phenomenon that is a key step in gene activation in response to numerous signaling pathways (Day et al, 2016). A number of techniques have been developed to assess impediments to RNAPII elongation (reviewed in Core & Adelman, 2019), it would be of key interest to examine the global mechanism of TRIM24 gene regulation. We have shown that TRIM24 is required to recruit CDK9 to the HIV-1 LTR. As such, a starting point for this global investigation would be to compare the transcriptome of TRIM24 null cells to ChIP-seq data of TRIM24 and CDK9. We have shown that TRIM24 regulates T cell activation to a degree, however, TRIM24 has been shown to be a strong driver of cancers including of breast (Ma et al, 2016) (Pathiraja et al, 2015) (Shah et al, 2021) 157 (Tsai et al, 2010). Perhaps it would be more fascinating to examine TRIM24 mediated transcriptional elongation within a cancer model such as the previously established models for breast or prostate cancers. 6.2 TRIM24 can be targeted by bromodomain inhibitors to reactivate latent HIV-1 Because TRIM24 is required for stimulation of transcription from the HIV-1 LTR, we were surprised to find that IACS-9571, a specific inhibitor of the TRIM24 C-terminal bromodomain, induces HIV-1 provirus expression in otherwise untreated cells. Additionally, treatment with this TRIM24 bromodomain inhibitor encourages productive HIV-1 expression in newly infected cells and inhibits formation of immediate latent repressed provirus. IACS-9571 synergizes with several LRAs to activate HIV-1 expression. Furthermore, co-treatment of CD4+ T cells from individuals with HIV-1 on antiretroviral therapy (ART) with PEP005 and IACS-9571 caused robust provirus expression. Notably, IACS-9571 did not cause global activation of T cells; rather, it inhibited induction of IL2 and CD69 expression in human PBMCs and Jurkat T cells treated with PEP005 or PMA. Here, we identified the bromodomain inhibitor IACS-9571 as novel HIV-1 latency reversing agent that unlike other compounds with this activity, causes partial suppression of T cell activation while inducing expression of latent provirus. Ideally, the LRA activity of IACS-9571 would next be assessed using an animal model. SIV-infected macaques are used to study HIV pathogenesis, but the use of bone marrow, liver, and thymus (BLT) humanized mice is much more common. The key experiment would involve exposing BLT humanized mouse to HIV-1 and subsequently administering ART and either a vehicle control or IACS-9571. At various later time points (on the scale of weeks to months), PBMCs be collected from the mice and the number of integrated HIV-1 proviruses would be estimated. The desired result would be that mice treated with IACS-9571 display a decrease in HIV-1 infected cells. Similar to our study (Horvath et al, 2023), HIV-1 infected mice could be administered IACS-9571 in combination with mechanistically distinct LRAs. A successful animal model study as described would give credence to a HIV-1 patient clinical trial. 6.3 Role of the Upstream Stimulatory factors for HIV-1 expression and the T cell inflammatory response Disruption of RBE3 and/or RBE1 prevents induction of proviral expression in response to T cell signaling, yet the function(s) of USF1 and USF2 remain unknown. In this Chapter, we identified the possibility that certain families of genes depend on particular combinations of the USFs: (i) USF1 or USF2 expressed adequately are sufficient to mediate transcription, (ii) USF1/USF2 heterodimers are essential, (iii) USF1 or USF2 homodimers are required. HIV-1 behaves as in (i) as we observed that USF1 was not required for expression of HIV-1, yet exogenous expression of either USF1 or USF2 was able to rescue expression in USF2 KO cells. This implies that USF2 depletion, along with the corresponding decrease in USF1 protein levels, caused LTR activation to be limited. Our RNA-seq analysis identified genes that are regulated analogous to HIV-1 as well as genes whose regulation is distinct from the virus. Those genes that 158 were differentially regulated upon USF2 ablation but not USF1 mirror that of HIV-1. Genes that were similarly affected by knockout of either USF1 or USF2 suggest that they require heterodimerization (ii), while those that were affected by USF1 deletion but not USF2 require homodimers (iii). Further work is required to solidify these notions. For instance, a gene may possess multiple regulatory elements capable of binding distinct populations of USFs. This may be mediated by combinatorial recruitment with other transcription factors such as TFII-I. Additionally, we can not determine genes in our RNA-seq dataset that are exclusively dependent upon USF2 as this factor’s expression is coupled to USF1. Reintroduction of USF1 in USF2 null cells would be required to begin this examination. Additionally, we identified USF1 and USF2 as key factors for regulating the T cell inflammatory response. HIV-1 preferentially infects activated T cells and here we observed that USF2 deletion causes these cells to be resistant to infection. Interestingly, in agreement with our work showing that USF1 and USF2 are proinflammatory factors, USF2 overexpression has been implicated in increased inflammation and resultant arthritis (Hu et al, 2020). Given this observation as well as the magnitude of effect that we found T cell activation to be disrupted, the USF factors must have greater physiological relevance. Inflammation is not always detrimental and is required to fend off viral infections. It is possible that in the absence of USFs, cells are susceptible to infection by RNA viruses such as influenza A or SARS-CoV2. Moreover, naïve T cells undergo various differentiation processes upon interaction with cytokines that stimulate activation pathways. Given our results, it would be expected that the USFs are required for proper T cell differentiation. USF1 and USF2 knockout mouse models have been generated and may be useful in this further exploration. 6.4 Chemical inhibitors of CDK8/19 enforce HIV-1 latency in CD4+ T cells In this Chapter we found that inhibitors of the Mediator complex kinases CDK8/19, Senexin A and BRD6989, inhibit induction of HIV-1 expression in response to latency reversing agents and T cell signaling agonists. These inhibitors were found to impair recruitment of RNAPII to the HIV-1 LTR. HIV-1 expression in response to several latency reversal agents was impaired upon disruption of CDK8 by shRNA or gene knockout. However, the effects of CDK8 depletion did not entirely mimic CDK8/19 kinase inhibition suggesting that the mediator kinases are not functionally redundant. Furthermore, treatment of CD4+ PBMCs isolated from people living with HIV-1 and who are receiving ART with Senexin A inhibited induction of virus replication in response to T cell stimulation by PMA and ionomycin. These observations indicate that the mediator kinases CDK8 and CDK19, play a significant role for regulation of HIV-1 transcription, and that small molecule inhibitors of these enzymes may contribute to therapies designed to promote deep latency involving the durable suppression of provirus expression. Likewise to what was discussed above regarding IACS-9571, BLT humanized mouse experiments would be the next step in this project. These mice would be exposed to HIV and then administered ART, a CDK8/19 inhibitor, or ART + CDK8/19 inhibitor. The gold standard 159 result would be if administration of the CDK8/19 inhibitor alone prevented HIV-1 progression similar to ART. Mouse models could also be examined over long periods of time to determine of CDK8/19 inhibition is capable of causing the latent reservoir to decay. 6.5 Closing remarks This thesis identified two complexes with HIV-1 therapeutic potential, RBF-2 and the Mediator kinase module. The TFII-I component of RBF-2 interacts with and recruits TRIM24 to the LTR to facilitate transcriptional elongation (Horvath et al, 2023). Inhibition of the TRIM24 bromodomain with IACS-9571 stimulates this process by increasing association of the co-factor with the viral promoter (Horvath et al, 2023). The presence of TRIM24 at the LTR correlates with enrichment of CDK9 and RNAPII Ser2-P, however, the mechanism(s) by which this occurs remain unknown (Fig. 6.1). I also assessed the role of the USF components of RBF-2 for the regulation of proviral latency, confirming these factors as activators. Interestingly, TFII-I was not needed for USF2 to bind the LTR while conversely, loss of USF2 prevented the association of TFII-I and TRIM24. This indicates that the USF factors serve as a platform for TFII-I/TRIM24 (Fig. 6.1), although additional regulatory roles are likely to exist. Finally, I identified the Mediator kinases CDK8/CDK19 as co-factors that potentiate HIV-1 expression. Inhibition of CDK8/19 activity limited recruitment of RNAPII to the LTR, though a possible role for transcriptional elongation cannot be overlooked. Moreover, I have not identified targets of CDK8/19 that potentiate HIV-1 expression (Fig. 6.1). CDK8/19 kinase activity has been shown to elevate expression of NFκB target genes (Chen et al, 2017) (Li et al, 2019), however, distinct activating NFκB phosphorylation sites have not been reported. Given that NFκB strongly potentiates HIV-1 expression in response to T cell activation (Sadowski & Mitchell, 2005), it is a prime candidate for examination. My thesis identified that RBF-2 activates HIV-1 expression, and interestingly, the activity of every component of the complex is regulated by phosphorylation (Fraser et al, 1998) (Horbach et al, 2015) (Roy, 2012). Moreover, USF1, USF2, and TFII-I are extensively phosphorylated upon T cell activation (Chen et al, 2005). This raises the intriguing notion that components of RBF-2 may be modified by CDK8/19 kinase activity to facilitate latency reversal. Hopefully the work presented in this thesis will serve as a node of information for which others can build off of to expand our knowledge of transcriptional regulation and to generate effective HIV-1 therapies. 160 Figure 6.1 Concluding graphical abstract. The USF factors serve as a platform for TFII-I and TRIM24. TRIM24 stimulates transcriptional elongation by facilitating recruitment of CDK9 to the viral promoter resulting in Ser-2 phosphorylation of the RNAPII CTD. The TRIM24 bromodomain inhibiter IACS-9571 is a latency reversal agent that increases LTR associated TRIM24. CDK8/19 kinase inhibitors (Senexin A, BRD6989) disrupt recruitment of RNAPII to the LTR, enforcing viral latency. 161 References Abbas, W. & Herbein, G. T-Cell Signaling in HIV-1 Infection. Open Virol J 7, 57–71 (2013). Aboelmagd, A., El Rayes, S. M., Gomaa, M. S., Fathalla, W., Ali, I. A. I., Nafie, M. S., Pottoo, F. H., Khan, F. A. & Ibrahim, M. M. Synthesis and Cytotoxic Activity of Novel Metal Complexes Derived from Methyl-3-(4-chlorophenyl)-3-hydroxy-2,2-dimethylpropanoate as Potential CDK8 Kinase Inhibitors. ACS Omega 6, 5244–5254 (2021). Acchioni, C., Remoli, A. L., Marsili, G., Acchioni, M., Nardolillo, I., Orsatti, R., Farcomeni, S., Palermo, E., Perrotti, E., Barreca, M. L., Sabatini, S., Sandini, S., Parolin, C., Lin, R., Borsetti, A., Hiscott, J. & Sgarbanti, M. Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus. J Virol 93, e00495-19 (2019). Afgan, E., Baker, D., Batut, B., van den Beek, M., Bouvier, D., Cech, M., Chilton, J., Clements, D., Coraor, N., Grüning, B. A., Guerler, A., Hillman-Jackson, J., Hiltemann, S., Jalili, V., Rasche, H., Soranzo, N., Goecks, J., Taylor, J., Nekrutenko, A. & Blankenberg, D. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46, W537–W544 (2018). Aiken, C. & Rousso, I. The HIV-1 capsid and reverse transcription. Retrovirology 18, 29 (2021). Ait-Ammar, A., Bellefroid, M., Daouad, F., Martinelli, V., Van Assche, J., Wallet, C., Rodari, A., De Rovere, M., Fahrenkrog, B., Schwartz, C., Van Lint, C., Gautier, V. & Rohr, O. Inhibition of HIV-1 gene transcription by KAP1 in myeloid lineage. Sci Rep 11, 2692 (2021). Albelda, S. M., Smith, C. W. & Ward, P. A. Adhesion molecules and inflammatory injury. FASEB J 8, 504–512 (1994). Allers, K., Hütter, G., Hofmann, J., Loddenkemper, C., Rieger, K., Thiel, E. & Schneider, T. Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood 117, 2791–2799 (2011). Allers, K. & Schneider, T. CCR5Δ32 mutation and HIV infection: basis for curative HIV therapy. Curr Opin Virol 14, 24–29 (2015). Allton, K., Jain, A. K., Herz, H.-M., Tsai, W.-W., Jung, S. Y., Qin, J., Bergmann, A., Johnson, R. L. & Barton, M. C. Trim24 targets endogenous p53 for degradation. Proc Natl Acad Sci U S A 106, 11612–11616 (2009). Andrews, G. K., Lee, D. K., Ravindra, R., Lichtlen, P., Sirito, M., Sawadogo, M. & Schaffner, W. The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development. EMBO J 20, 1114–1122 (2001). 162 Aoki, S., Watanabe, Y., Sanagawa, M., Setiawan, A., Kotoku, N. & Kobayashi, M. Cortistatins A, B, C, and D, anti-angiogenic steroidal alkaloids, from the marine sponge Corticium simplex. J Am Chem Soc 128, 3148–3149 (2006). Appikonda, S., Thakkar, K. N. & Barton, M. C. Regulation of gene expression in human cancers by TRIM24. Drug Discov Today Technol 19, 57–63 (2016). Appikonda, S., Thakkar, K. N., Shah, P. K., Dent, S. Y. R., Andersen, J. N. & Barton, M. C. Cross-talk between chromatin acetylation and SUMOylation of tripartite motif-containing protein 24 (TRIM24) impacts cell adhesion. J Biol Chem 293, 7476–7485 (2018). Archin, N. M., Liberty, A. L., Kashuba, A. D., Choudhary, S. K., Kuruc, J. D., Crooks, A. M., Parker, D. C., Anderson, E. M., Kearney, M. F., Strain, M. C., Richman, D. D., Hudgens, M. G., Bosch, R. J., Coffin, J. M., Eron, J. J., Hazuda, D. J. & Margolis, D. M. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487, 482–485 (2012). Arhel, N. J., Souquere-Besse, S., Munier, S., Souque, P., Guadagnini, S., Rutherford, S., Prévost, M.-C., Allen, T. D. & Charneau, P. HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J 26, 3025–3037 (2007). Aristizabal, M. J., Negri, G. L., Benschop, J. J., Holstege, F. C. P., Krogan, N. J. & Kobor, M. S. High-throughput genetic and gene expression analysis of the RNAPII-CTD reveals unexpected connections to SRB10/CDK8. PLoS Genet 9, e1003758 (2013). Ashe, M. P., Furger, A. & Proudfoot, N. J. Stem-loop 1 of the U1 snRNP plays a critical role in the suppression of HIV-1 polyadenylation. RNA 6, 170–177 (2000). Ashe, M. P., Griffin, P., James, W. & Proudfoot, N. J. Poly(A) site selection in the HIV-1 provirus: inhibition of promoter-proximal polyadenylation by the downstream major splice donor site. Genes Dev 9, 3008–3025 (1995). Bailey, J. R., Sedaghat, A. R., Kieffer, T., Brennan, T., Lee, P. K., Wind-Rotolo, M., Haggerty, C. M., Kamireddi, A. R., Liu, Y., Lee, J., Persaud, D., Gallant, J. E., Cofrancesco, J., Quinn, T. C., Wilke, C. O., Ray, S. C., Siliciano, J. D., Nettles, R. E. & Siliciano, R. F. Residual human immunodeficiency virus type 1 viremia in some patients on antiretroviral therapy is dominated by a small number of invariant clones rarely found in circulating CD4+ T cells. J Virol 80, 6441–6457 (2006). Bancerek, J., Poss, Z. C., Steinparzer, I., Sedlyarov, V., Pfaffenwimmer, T., Mikulic, I., Dölken, L., Strobl, B., Müller, M., Taatjes, D. J. & Kovarik, P. CDK8 kinase phosphorylates transcription factor STAT1 to selectively regulate the interferon response. Immunity 38, 250–262 (2013). 163 Barat, C., Lullien, V., Schatz, O., Keith, G., Nugeyre, M. T., Grüninger-Leitch, F., Barré-Sinoussi, F., LeGrice, S. F. & Darlix, J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J 8, 3279–3285 (1989). Barbian, H. J., Seaton, M. S., Narasipura, S. D., Wallace, J., Rajan, R., Sha, B. E. & Al-Harthi, L. β-catenin regulates HIV latency and modulates HIV reactivation. PLoS Pathog 18, e1010354 (2022). Battivelli, E., Dahabieh, M. S., Abdel-Mohsen, M., Svensson, J. P., Tojal Da Silva, I., Cohn, L. B., Gramatica, A., Deeks, S., Greene, W. C., Pillai, S. K. & Verdin, E. Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells. Elife 7, e34655 (2018). Battivelli, E. & Verdin, E. HIVGKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4+ T Cells. Bio Protoc 8, e3050 (2018). Baxevanis, A. D. & Vinson, C. R. Interactions of coiled coils in transcription factors: where is the specificity? Curr Opin Genet Dev 3, 278–285 (1993). Bayarsaihan, D., Makeyev, A. V. & Enkhmandakh, B. Epigenetic modulation by TFII-I during embryonic stem cell differentiation. J Cell Biochem 113, 3056–3060 (2012). Bell, B. & Sadowski, I. Ras-responsiveness of the HIV-1 LTR requires RBF-1 and RBF-2 binding sites. Oncogene 13, 2687–2697 (1996). Berkhout, B., Silverman, R. H. & Jeang, K. T. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 59, 273–282 (1989). Bernhard, W., Barreto, K., Raithatha, S. & Sadowski, I. An upstream YY1 binding site on the HIV-1 LTR contributes to latent infection. PLoS One 8, e77052 (2013). Bernhard, W., Barreto, K., Saunders, A., Dahabieh, M. S., Johnson, P. & Sadowski, I. The Suv39H1 methyltransferase inhibitor chaetocin causes induction of integrated HIV-1 without producing a T cell response. FEBS Lett 585, 3549–3554 (2011). Besnard, E., Hakre, S., Kampmann, M., Lim, H. W., Hosmane, N. N., Martin, A., Bassik, M. C., Verschueren, E., Battivelli, E., Chan, J., Svensson, J. P., Gramatica, A., Conrad, R. J., Ott, M., Greene, W. C., Krogan, N. J., Siliciano, R. F., Weissman, J. S. & Verdin, E. The mTOR Complex Controls HIV Latency. Cell Host Microbe 20, 785–797 (2016). Bieniasz, P. D., Grdina, T. A., Bogerd, H. P. & Cullen, B. R. Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Proc Natl Acad Sci U S A 96, 7791–7796 (1999). 164 Blazkova, J., Trejbalova, K., Gondois-Rey, F., Halfon, P., Philibert, P., Guiguen, A., Verdin, E., Olive, D., Van Lint, C., Hejnar, J. & Hirsch, I. CpG methylation controls reactivation of HIV from latency. PLoS Pathog 5, e1000554 (2009). Bosque, A., Nilson, K. A., Macedo, A. B., Spivak, A. M., Archin, N. M., Van Wagoner, R. M., Martins, L. J., Novis, C. L., Szaniawski, M. A., Ireland, C. M., Margolis, D. M., Price, D. H. & Planelles, V. Benzotriazoles Reactivate Latent HIV-1 through Inactivation of STAT5 SUMOylation. Cell Rep 18, 1324–1334 (2017). Bouafia, A., Corre, S., Gilot, D., Mouchet, N., Prince, S. & Galibert, M.-D. p53 requires the stress sensor USF1 to direct appropriate cell fate decision. PLoS Genet 10, e1004309 (2014). Bourgeois, C. F., Kim, Y. K., Churcher, M. J., West, M. J. & Karn, J. Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences. Mol Cell Biol 22, 1079–1093 (2002). Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N. & Ting, A. Y. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol 36, 880–887 (2018). Briggs, J. A. G., Wilk, T., Welker, R., Kräusslich, H.-G. & Fuller, S. D. Structural organization of authentic, mature HIV-1 virions and cores. EMBO J 22, 1707–1715 (2003). Brooks, D. G., Arlen, P. A., Gao, L., Kitchen, C. M. R. & Zack, J. A. Identification of T cell-signaling pathways that stimulate latent HIV in primary cells. Proc Natl Acad Sci U S A 100, 12955–12960 (2003). Bruner, K. M., Murray, A. J., Pollack, R. A., Soliman, M. G., Laskey, S. B., Capoferri, A. A., Lai, J., Strain, M. C., Lada, S. M., Hoh, R., Ho, Y.-C., Richman, D. D., Deeks, S. G., Siliciano, J. D. & Siliciano, R. F. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 22, 1043–1049 (2016). Budhiraja, S., Famiglietti, M., Bosque, A., Planelles, V. & Rice, A. P. Cyclin T1 and CDK9 T-loop phosphorylation are downregulated during establishment of HIV-1 latency in primary resting memory CD4+ T cells. J Virol 87, 1211–1220 (2013). Bunch, H., Lawney, B. P., Lin, Y.-F., Asaithamby, A., Murshid, A., Wang, Y. E., Chen, B. P. C. & Calderwood, S. K. Transcriptional elongation requires DNA break-induced signalling. Nat Commun 6, 10191 (2015). Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol Cell 36, 541–546 (2009). Buzon, V., Natrajan, G., Schibli, D., Campelo, F., Kozlov, M. M. & Weissenhorn, W. Crystal structure of HIV-1 gp41 including both fusion peptide and membrane proximal external regions. PLoS Pathog 6, e1000880 (2010). 165 Caputo, A., Sodroski, J. G. & Haseltine, W. A. Constitutive expression of HIV-1 tat protein in human Jurkat T cells using a BK virus vector. J Acquir Immune Defic Syndr (1988) 3, 372–379 (1990). Carthew, R. W., Chodosh, L. A. & Sharp, P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell 43, 439–448 (1985). Cary, D. C., Rheinberger, M., Rojc, A. & Peterlin, B. M. HIV Transcription Is Independent of Mediator Kinases. AIDS Res Hum Retroviruses 35, 710–717 (2019). Cee, V. J., Chen, D. Y.-K., Lee, M. R. & Nicolaou, K. C. Cortistatin A is a high-affinity ligand of protein kinases ROCK, CDK8, and CDK11. Angew Chem Int Ed Engl 48, 8952–8957 (2009). Chan, D. C., Fass, D., Berger, J. M. & Kim, P. S. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263–273 (1997). Charneau, P., Alizon, M. & Clavel, F. A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol 66, 2814–2820 (1992). Chen, H.-C., Martinez, J. P., Zorita, E., Meyerhans, A. & Filion, G. J. Position effects influence HIV latency reversal. Nat Struct Mol Biol 24, 47–54 (2017). Chen, J., Nikolaitchik, O., Singh, J., Wright, A., Bencsics, C. E., Coffin, J. M., Ni, N., Lockett, S., Pathak, V. K. & Hu, W.-S. High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis. Proc Natl Acad Sci U S A 106, 13535–13540 (2009). Chen, J., Malcolm, T., Estable, M. C., Roeder, R. G. & Sadowski, I. TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type 1 long terminal repeat in cooperation with USF. J Virol 79, 4396–4406 (2005). Chen, M., Li, J., Liang, J., Thompson, Z. S., Kathrein, K., Broude, E. V. & Roninson, I. B. Systemic Toxicity Reported for CDK8/19 Inhibitors CCT251921 and MSC2530818 Is Not Due to Target Inhibition. Cells 8, 1413 (2019). Chen, M., Liang, J., Ji, H., Yang, Z., Altilia, S., Hu, B., Schronce, A., McDermott, M. S. J., Schools, G. P., Lim, C.-U., Oliver, D., Shtutman, M. S., Lu, T., Stark, G. R., Porter, D. C., Broude, E. V. & Roninson, I. B. CDK8/19 Mediator kinases potentiate induction of transcription by NFκB. Proc Natl Acad Sci U S A 114, 10208–10213 (2017). Cheung, E., Mayr, P., Coda-Zabetta, F., Woodman, P. G. & Boam, D. S. DNA-binding activity of the transcription factor upstream stimulatory factor 1 (USF-1) is regulated by cyclin-dependent phosphorylation. Biochem J 344 Pt 1, 145–152 (1999). 166 Chi, T. F., Horbach, T., Götz, C., Kietzmann, T. & Dimova, E. Y. Cyclin-Dependent Kinase 5 (CDK5)-Mediated Phosphorylation of Upstream Stimulatory Factor 2 (USF2) Contributes to Carcinogenesis. Cancers (Basel) 11, 523 (2019). Chong, L., Shi, X. & Zhu, Y. Signal Integration by Cyclin-Dependent Kinase 8 (CDK8) Module and Other Mediator Subunits in Biotic and Abiotic Stress Responses. Int J Mol Sci 22, 354 (2020). Chun, T. W., Davey, R. T., Ostrowski, M., Shawn Justement, J., Engel, D., Mullins, J. I. & Fauci, A. S. Relationship between pre-existing viral reservoirs and the re-emergence of plasma viremia after discontinuation of highly active anti-retroviral therapy. Nat Med 6, 757–761 (2000). Clavel, F., Guétard, D., Brun-Vézinet, F., Chamaret, S., Rey, M. A., Santos-Ferreira, M. O., Laurent, A. G., Dauguet, C., Katlama, C. & Rouzioux, C. Isolation of a new human retrovirus from West African patients with AIDS. Science 233, 343–346 (1986). Coffin, J. M., Hughes, S. H. & Varmus, H. E. in Retroviruses (eds. Coffin, J. M., Hughes, S. H. & Varmus, H. E.) (Cold Spring Harbor Laboratory Press, 1997). at <http://www.ncbi.nlm.nih.gov/books/NBK19465/> Cohn, L. B., Chomont, N. & Deeks, S. G. The Biology of the HIV-1 Latent Reservoir and Implications for Cure Strategies. Cell Host Microbe 27, 519–530 (2020). Collins, A., Savas, J. & Doerfler, L. Nonsurgical Treatments for Nonmelanoma Skin Cancer. Dermatol Clin 37, 435–441 (2019). Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S. & Landau, N. R. Change in coreceptor use correlates with disease progression in HIV-1--infected individuals. J Exp Med 185, 621–628 (1997). Conrad, R. J., Fozouni, P., Thomas, S., Sy, H., Zhang, Q., Zhou, M.-M. & Ott, M. The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes. Mol Cell 67, 1001-1012.e6 (2017). Contreras, X., Schweneker, M., Chen, C.-S., McCune, J. M., Deeks, S. G., Martin, J. & Peterlin, B. M. Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells. J Biol Chem 284, 6782–6789 (2009). Core, L. & Adelman, K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes Dev 33, 960–982 (2019). Corre, S., Primot, A., Sviderskaya, E., Bennett, D. C., Vaulont, S., Goding, C. R. & Galibert, M.-D. UV-induced expression of key component of the tanning process, the POMC and MC1R genes, is dependent on the p-38-activated upstream stimulating factor-1 (USF-1). J Biol Chem 279, 51226–51233 (2004). 167 Cosnefroy, O., Murray, P. J. & Bishop, K. N. HIV-1 capsid uncoating initiates after the first strand transfer of reverse transcription. Retrovirology 13, 58 (2016). Coull, J. J., Romerio, F., Sun, J. M., Volker, J. L., Galvin, K. M., Davie, J. R., Shi, Y., Hansen, U. & Margolis, D. M. The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1. J Virol 74, 6790–6799 (2000). Courtney, A. H., Lo, W.-L. & Weiss, A. TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci 43, 108–123 (2018). Cullen, B. R., Lomedico, P. T. & Ju, G. Transcriptional interference in avian retroviruses--implications for the promoter insertion model of leukaemogenesis. Nature 307, 241–245 (1984). d’Adda di Fagagna, F., Marzio, G., Gutierrez, M. I., Kang, L. Y., Falaschi, A. & Giacca, M. Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1. J Virol 69, 2765–2775 (1995). Dahabieh, M. S., Ooms, M., Brumme, C., Taylor, J., Harrigan, P. R., Simon, V. & Sadowski, I. Direct non-productive HIV-1 infection in a T-cell line is driven by cellular activation state and NFκB. Retrovirology 11, 17 (2014). Dahabieh, M. S., Ooms, M., Malcolm, T., Simon, V. & Sadowski, I. Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter. Virology 418, 57–66 (2011). Dahabieh, M. S., Ooms, M., Simon, V. & Sadowski, I. A doubly fluorescent HIV-1 reporter shows that the majority of integrated HIV-1 is latent shortly after infection. J Virol 87, 4716–4727 (2013). Das, A. T., Klaver, B. & Berkhout, B. A hairpin structure in the R region of the human immunodeficiency virus type 1 RNA genome is instrumental in polyadenylation site selection. J Virol 73, 81–91 (1999). Day, D. S., Zhang, B., Stevens, S. M., Ferrari, F., Larschan, E. N., Park, P. J. & Pu, W. T. Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types. Genome Biol 17, 120 (2016). Deeks, S. G. & Walker, B. D. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 27, 406–416 (2007). Demont, E. H., Bamborough, P., Chung, C.-W., Craggs, P. D., Fallon, D., Gordon, L. J., Grandi, P., Hobbs, C. I., Hussain, J., Jones, E. J., Le Gall, A., Michon, A.-M., Mitchell, D. J., Prinjha, R. K., Roberts, A. D., Sheppard, R. J. & Watson, R. J. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. ACS Med Chem Lett 5, 1190–1195 (2014). 168 Desimio, M. G., Giuliani, E. & Doria, M. The histone deacetylase inhibitor SAHA simultaneously reactivates HIV-1 from latency and up-regulates NKG2D ligands sensitizing for natural killer cell cytotoxicity. Virology 510, 9–21 (2017). Dobersch, S., Rubio, K., Singh, I., Günther, S., Graumann, J., Cordero, J., Castillo-Negrete, R., Huynh, M. B., Mehta, A., Braubach, P., Cabrera-Fuentes, H., Bernhagen, J., Chao, C.-M., Bellusci, S., Günther, A., Preissner, K. T., Kugel, S., Dobreva, G., Wygrecka, M., Braun, T., Papy-Garcia, D. & Barreto, G. Positioning of nucleosomes containing γ-H2AX precedes active DNA demethylation and transcription initiation. Nat Commun 12, 1072 (2021). Doi-Katayama, Y., Hayashi, F., Inoue, M., Yabuki, T., Aoki, M., Seki, E., Matsuda, T., Kigawa, T., Yoshida, M., Shirouzu, M., Terada, T., Hayashizaki, Y., Yokoyama, S. & Hirota, H. Solution structure of the general transcription factor 2I domain in mouse TFII-I protein. Protein Sci 16, 1788–1792 (2007). Douglass, A. D. & Vale, R. D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005). du Chéné, I., Basyuk, E., Lin, Y.-L., Triboulet, R., Knezevich, A., Chable-Bessia, C., Mettling, C., Baillat, V., Reynes, J., Corbeau, P., Bertrand, E., Marcello, A., Emiliani, S., Kiernan, R. & Benkirane, M. Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency. EMBO J 26, 424–435 (2007). Du, H., Roy, A. L. & Roeder, R. G. Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters. EMBO J 12, 501–511 (1993). Dutta, D., Barr, V. A., Akpan, I., Mittelstadt, P. R., Singha, L. I., Samelson, L. E. & Ashwell, J. D. Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol 18, 196–204 (2017). el Kharroubi, A. & Verdin, E. Protein-DNA interactions within DNase I-hypersensitive sites located downstream of the HIV-1 promoter. J Biol Chem 269, 19916–19924 (1994). Elliott, J. H., McMahon, J. H., Chang, C. C., Lee, S. A., Hartogensis, W., Bumpus, N., Savic, R., Roney, J., Hoh, R., Solomon, A., Piatak, M., Gorelick, R. J., Lifson, J., Bacchetti, P., Deeks, S. G. & Lewin, S. R. Short-term administration of disulfiram for reversal of latent HIV infection: a phase 2 dose-escalation study. Lancet HIV 2, e520-529 (2015). Elliott, J. H., Wightman, F., Solomon, A., Ghneim, K., Ahlers, J., Cameron, M. J., Smith, M. Z., Spelman, T., McMahon, J., Velayudham, P., Brown, G., Roney, J., Watson, J., Prince, M. H., Hoy, J. F., Chomont, N., Fromentin, R., Procopio, F. A., Zeidan, J., Palmer, S., Odevall, L., Johnstone, R. W., Martin, B. P., Sinclair, E., Deeks, S. G., Hazuda, D. J., Cameron, P. U., 169 Sékaly, R.-P. & Lewin, S. R. Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathog 10, e1004473 (2014). Emiliani, S., Fischle, W., Ott, M., Van Lint, C., Amella, C. A. & Verdin, E. Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol 72, 1666–1670 (1998). Emiliani, S., Van Lint, C., Fischle, W., Paras, P., Ott, M., Brady, J. & Verdin, E. A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency. Proc Natl Acad Sci U S A 93, 6377–6381 (1996). ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012). Estable, M. C., Bell, B., Merzouki, A., Montaner, J. S., O’Shaughnessy, M. V. & Sadowski, I. J. Human immunodeficiency virus type 1 long terminal repeat variants from 42 patients representing all stages of infection display a wide range of sequence polymorphism and transcription activity. J Virol 70, 4053–4062 (1996). Estable, M. C., Hirst, M., Bell, B., O’Shaughnessy, M. V. & Sadowski, I. Purification of RBF-2, a transcription factor with specificity for the most conserved cis-element of naturally occurring HIV-1 LTRs. J Biomed Sci 6, 320–332 (1999). Fant, C. B. & Taatjes, D. J. Regulatory functions of the Mediator kinases CDK8 and CDK19. Transcription 10, 76–90 (2019). Farnet, C. M. & Bushman, F. D. HIV-1 cDNA integration: requirement of HMG I(Y) protein for function of preintegration complexes in vitro. Cell 88, 483–492 (1997). Farnet, C. M. & Haseltine, W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol 65, 1910–1915 (1991). Fassati, A. & Goff, S. P. Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1. J Virol 75, 3626–3635 (2001). Finzi, D., Hermankova, M., Pierson, T., Carruth, L. M., Buck, C., Chaisson, R. E., Quinn, T. C., Chadwick, K., Margolick, J., Brookmeyer, R., Gallant, J., Markowitz, M., Ho, D. D., Richman, D. D. & Siliciano, R. F. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278, 1295–1300 (1997). Firestein, R., Bass, A. J., Kim, S. Y., Dunn, I. F., Silver, S. J., Guney, I., Freed, E., Ligon, A. H., Vena, N., Ogino, S., Chheda, M. G., Tamayo, P., Finn, S., Shrestha, Y., Boehm, J. S., Jain, S., Bojarski, E., Mermel, C., Barretina, J., Chan, J. A., Baselga, J., Tabernero, J., Root, D. E., Fuchs, C. S., Loda, M., Shivdasani, R. A., Meyerson, M. & Hahn, W. C. CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature 455, 547–551 (2008). 170 Fischer, D. K., Saito, A., Kline, C., Cohen, R., Watkins, S. C., Yamashita, M. & Ambrose, Z. CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes. J Virol 93, e00214-19 (2019). Fischl, M. A., Richman, D. D., Grieco, M. H., Gottlieb, M. S., Volberding, P. A., Laskin, O. L., Leedom, J. M., Groopman, J. E., Mildvan, D. & Schooley, R. T. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. A double-blind, placebo-controlled trial. N Engl J Med 317, 185–191 (1987). Fong, K.-W., Zhao, J. C., Song, B., Zheng, B. & Yu, J. TRIM28 protects TRIM24 from SPOP-mediated degradation and promotes prostate cancer progression. Nat Commun 9, 5007 (2018). Forshey, B. M., von Schwedler, U., Sundquist, W. I. & Aiken, C. Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol 76, 5667–5677 (2002). Francis, A. C., Marin, M., Shi, J., Aiken, C. & Melikyan, G. B. Time-Resolved Imaging of Single HIV-1 Uncoating In Vitro and in Living Cells. PLoS Pathog 12, e1005709 (2016). Frankel, A. D. & Young, J. A. HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67, 1–25 (1998). Fraser, R. A., Heard, D. J., Adam, S., Lavigne, A. C., Le Douarin, B., Tora, L., Losson, R., Rochette-Egly, C. & Chambon, P. The putative cofactor TIF1alpha is a protein kinase that is hyperphosphorylated upon interaction with liganded nuclear receptors. J Biol Chem 273, 16199–16204 (1998). Friedman, J., Cho, W.-K., Chu, C. K., Keedy, K. S., Archin, N. M., Margolis, D. M. & Karn, J. Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2. J Virol 85, 9078–9089 (2011). Friedman, J., Cho, W.-K., Chu, C. K., Keedy, K. S., Archin, N. M., Margolis, D. M. & Karn, J. Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2. J Virol 85, 9078–9089 (2011). Fryer, C. J., White, J. B. & Jones, K. A. Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16, 509–520 (2004). Fujinaga, K., Irwin, D., Huang, Y., Taube, R., Kurosu, T. & Peterlin, B. M. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol 24, 787–795 (2004). Galibert, M. D., Boucontet, L., Goding, C. R. & Meo, T. Recognition of the E-C4 element from the C4 complement gene promoter by the upstream stimulatory factor-1 transcription factor. J Immunol 159, 6176–6183 (1997). 171 Galibert, M. D., Carreira, S. & Goding, C. R. The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression. EMBO J 20, 5022–5031 (2001). Gechijian, L. N., Buckley, D. L., Lawlor, M. A., Reyes, J. M., Paulk, J., Ott, C. J., Winter, G. E., Erb, M. A., Scott, T. G., Xu, M., Seo, H.-S., Dhe-Paganon, S., Kwiatkowski, N. P., Perry, J. A., Qi, J., Gray, N. S. & Bradner, J. E. Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands. Nat Chem Biol 14, 405–412 (2018). Geis, F. K. & Goff, S. P. Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced. Proc Natl Acad Sci U S A 116, 23735–23742 (2019). Gelderblom, H. R., Ozel, M. & Pauli, G. Morphogenesis and morphology of HIV. Structure-function relations. Arch Virol 106, 1–13 (1989). Gilmartin, G. M., Fleming, E. S., Oetjen, J. & Graveley, B. R. CPSF recognition of an HIV-1 mRNA 3’-processing enhancer: multiple sequence contacts involved in poly(A) site definition. Genes Dev 9, 72–83 (1995). Gold, M. O., Tassan, J. P., Nigg, E. A., Rice, A. P. & Herrmann, C. H. Viral transactivators E1A and VP16 interact with a large complex that is associated with CTD kinase activity and contains CDK8. Nucleic Acids Res 24, 3771–3777 (1996). Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999). Gregor, P. D., Sawadogo, M. & Roeder, R. G. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev 4, 1730–1740 (1990). Groenen, P. M., Garcia, E., Debeer, P., Devriendt, K., Fryns, J. P. & Van de Ven, W. J. Structure, sequence, and chromosome 19 localization of human USF2 and its rearrangement in a patient with multicystic renal dysplasia. Genomics 38, 141–148 (1996). Groner, A. C., Cato, L., de Tribolet-Hardy, J., Bernasocchi, T., Janouskova, H., Melchers, D., Houtman, R., Cato, A. C. B., Tschopp, P., Gu, L., Corsinotti, A., Zhong, Q., Fankhauser, C., Fritz, C., Poyet, C., Wagner, U., Guo, T., Aebersold, R., Garraway, L. A., Wild, P. J., Theurillat, J.-P. & Brown, M. TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer. Cancer Cell 29, 846–858 (2016). Gu, W., Wang, C., Li, W., Hsu, F.-N., Tian, L., Zhou, J., Yuan, C., Xie, X.-J., Jiang, T., Addya, S., Tai, Y., Kong, B. & Ji, J.-Y. Tumor-suppressive effects of CDK8 in endometrial cancer cells. Cell Cycle 12, 987–999 (2013). 172 Gu, Y., Zuo, X., Zhang, S., Ouyang, Z., Jiang, S., Wang, F. & Wang, G. The Mechanism behind Influenza Virus Cytokine Storm. Viruses 13, 1362 (2021). Gupta, R. K., Abdul-Jawad, S., McCoy, L. E., Mok, H. P., Peppa, D., Salgado, M., Martinez-Picado, J., Nijhuis, M., Wensing, A. M. J., Lee, H., Grant, P., Nastouli, E., Lambert, J., Pace, M., Salasc, F., Monit, C., Innes, A. J., Muir, L., Waters, L., Frater, J., Lever, A. M. L., Edwards, S. G., Gabriel, I. H. & Olavarria, E. HIV-1 remission following CCR5Δ32/Δ32 haematopoietic stem-cell transplantation. Nature 568, 244–248 (2019). Gutiérrez, C., Serrano-Villar, S., Madrid-Elena, N., Pérez-Elías, M. J., Martín, M. E., Barbas, C., Ruipérez, J., Muñoz, E., Muñoz-Fernández, M. A., Castor, T. & Moreno, S. Bryostatin-1 for latent virus reactivation in HIV-infected patients on antiretroviral therapy. AIDS 30, 1385–1392 (2016). Haddad, N., Weeks, A., Robert, A. & Totten, S. HIV in Canada-surveillance report, 2019. Can Commun Dis Rep 47, 77–86 (2021). Han, M. & Sun, Y. Pharmacological targeting of Tripartite Motif Containing 24 for the treatment of glioblastoma. J Transl Med 19, 505 (2021). Han, Y., Lin, Y. B., An, W., Xu, J., Yang, H.-C., O’Connell, K., Dordai, D., Boeke, J. D., Siliciano, J. D. & Siliciano, R. F. Orientation-dependent regulation of integrated HIV-1 expression by host gene transcriptional readthrough. Cell Host Microbe 4, 134–146 (2008). Hansen, M. M. K., Martin, B. & Weinberger, L. S. HIV Latency: Stochastic across Multiple Scales. Cell Host Microbe 26, 703–705 (2019). Hashemi, F. B., Barreto, K., Bernhard, W., Hashemi, P., Lomness, A. & Sadowski, I. HIV Provirus Stably Reproduces Parental Latent and Induced Transcription Phenotypes Regardless of the Chromosomal Integration Site. J Virol 90, 5302–5314 (2016). Hashemi, P., Barreto, K., Bernhard, W., Lomness, A., Honson, N., Pfeifer, T. A., Harrigan, P. R. & Sadowski, I. Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency. EMBO Mol Med 10, 160–174 (2018). Hashemi, P. & Sadowski, I. Diversity of small molecule HIV-1 latency reversing agents identified in low- and high-throughput small molecule screens. Med Res Rev 40, 881–908 (2020). Hatakeyama, S. TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis. Trends Biochem Sci 42, 297–311 (2017). He, G. & Margolis, D. M. Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat. Mol Cell Biol 22, 2965–2973 (2002). 173 Herquel, B., Ouararhni, K., Khetchoumian, K., Ignat, M., Teletin, M., Mark, M., Béchade, G., Van Dorsselaer, A., Sanglier-Cianférani, S., Hamiche, A., Cammas, F., Davidson, I. & Losson, R. Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma. Proc Natl Acad Sci U S A 108, 8212–8217 (2011). HHS. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV. (2022). at <https://clinicalinfo.hiv.gov/en/guidelines> Hill, A. L. Mathematical Models of HIV Latency. Curr Top Microbiol Immunol 417, 131–156 (2018). Hirst, M., Ho, C., Sabourin, L., Rudnicki, M., Penn, L. & Sadowski, I. A two-hybrid system for transactivator bait proteins. Proc Natl Acad Sci U S A 98, 8726–8731 (2001). Ho, D. D. Time to hit HIV, early and hard. N Engl J Med 333, 450–451 (1995). Ho, Y.-C., Shan, L., Hosmane, N. N., Wang, J., Laskey, S. B., Rosenbloom, D. I. S., Lai, J., Blankson, J. N., Siliciano, J. D. & Siliciano, R. F. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155, 540–551 (2013). Horbach, T., Götz, C., Kietzmann, T. & Dimova, E. Y. Protein kinases as switches for the function of upstream stimulatory factors: implications for tissue injury and cancer. Front Pharmacol 6, 3 (2015). Horvath, R. M., Brumme, Z. L. & Sadowski, I. Inhibition of the TRIM24 bromodomain reactivates latent HIV-1. Sci Rep 13, 556 (2023). Horvath, R. M., Dahabieh, M., Malcolm, T. & Sadowski, I. TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation. Commun Biol 6, 86 (2023). Horvath, R. M. & Sadowski, I. Upstream Stimulatory Factors Regulate HIV-1 Latency and Are Required for Robust T Cell Activation. Viruses 15, 1470 (2023). Hu, D., Tjon, E. C., Andersson, K. M., Molica, G. M., Pham, M. C., Healy, B., Murugaiyan, G., Pochet, N., Kuchroo, V. K., Bokarewa, M. I. & Weiner, H. L. Aberrant expression of USF2 in refractory rheumatoid arthritis and its regulation of proinflammatory cytokines in Th17 cells. Proc Natl Acad Sci U S A 117, 30639–30648 (2020). Hu, W.-S. & Hughes, S. H. HIV-1 reverse transcription. Cold Spring Harb Perspect Med 2, a006882 (2012). Huang, H., Liu, S., Jean, M., Simpson, S., Huang, H., Merkley, M., Hayashi, T., Kong, W., Rodríguez-Sánchez, I., Zhang, X., Yosief, H. O., Miao, H., Que, J., Kobie, J. J., Bradner, J., Santoso, N. G., Zhang, W. & Zhu, J. A Novel Bromodomain Inhibitor Reverses HIV-1 Latency 174 through Specific Binding with BRD4 to Promote Tat and P-TEFb Association. Front Microbiol 8, 1035 (2017). Huang, Y., Mak, J., Cao, Q., Li, Z., Wainberg, M. A. & Kleiman, L. Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1. J Virol 68, 7676–7683 (1994). Huang, Y., Shalom, A., Li, Z., Wang, J., Mak, J., Wainberg, M. A. & Kleiman, L. Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription. J Virol 70, 4700–4706 (1996). Hubert, J. B., Burgard, M., Dussaix, E., Tamalet, C., Deveau, C., Le Chenadec, J., Chaix, M. L., Marchadier, E., Vildé, J. L., Delfraissy, J. F., Meyer, L. & Rouzioux, null. Natural history of serum HIV-1 RNA levels in 330 patients with a known date of infection. The SEROCO Study Group. AIDS 14, 123–131 (2000). Hughes, A. & Corrah, T. Human immunodeficiency virus type 2 (HIV2). Blood Rev 4, 158–164 (1990). Hulme, A. E., Kelley, Z., Foley, D. & Hope, T. J. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells. J Virol 89, 5350–5361 (2015). Hulme, A. E., Perez, O. & Hope, T. J. Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci U S A 108, 9975–9980 (2011). Hungnes, O., Tjøtta, E. & Grinde, B. Mutations in the central polypurine tract of HIV-1 result in delayed replication. Virology 190, 440–442 (1992). Hütter, G. Stem cell transplantation in strategies for curing HIV/AIDS. AIDS Res Ther 13, 31 (2016). Hwang, S. S., Boyle, T. J., Lyerly, H. K. & Cullen, B. R. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253, 71–74 (1991). Imai, K., Togami, H. & Okamoto, T. Involvement of histone H3 lysine 9 (H3K9) methyltransferase G9a in the maintenance of HIV-1 latency and its reactivation by BIX01294. J Biol Chem 285, 16538–16545 (2010). Irwan, I. D., Bogerd, H. P. & Cullen, B. R. Epigenetic silencing by the SMC5/6 complex mediates HIV-1 latency. Nat Microbiol 7, 2101–2113 (2022). Isel, C., Ehresmann, C., Keith, G., Ehresmann, B. & Marquet, R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol 247, 236–250 (1995). 175 Isel, C. & Karn, J. Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation. J Mol Biol 290, 929–941 (1999). Ismail, P. M., Lu, T. & Sawadogo, M. Loss of USF transcriptional activity in breast cancer cell lines. Oncogene 18, 5582–5591 (1999). Ivanov, D., Kwak, Y. T., Guo, J. & Gaynor, R. B. Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol Cell Biol 20, 2970–2983 (2000). Jain, A. K., Allton, K., Duncan, A. D. & Barton, M. C. TRIM24 is a p53-induced E3-ubiquitin ligase that undergoes ATM-mediated phosphorylation and autodegradation during DNA damage. Mol Cell Biol 34, 2695–2709 (2014). Jeronimo, C., Langelier, M.-F., Bataille, A. R., Pascal, J. M., Pugh, B. F. & Robert, F. Tail and Kinase Modules Differently Regulate Core Mediator Recruitment and Function In Vivo. Mol Cell 64, 455–466 (2016). Jiang, G., Mendes, E. A., Kaiser, P., Wong, D. P., Tang, Y., Cai, I., Fenton, A., Melcher, G. P., Hildreth, J. E. K., Thompson, G. R., Wong, J. K. & Dandekar, S. Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation. PLoS Pathog 11, e1005066 (2015). Jiang, Y., Rubin, L., Peng, T., Liu, L., Xing, X., Lazarovici, P. & Zheng, W. Cytokine storm in COVID-19: from viral infection to immune responses, diagnosis and therapy. Int J Biol Sci 18, 459–472 (2022). Johannessen, L., Sundberg, T. B., O’Connell, D. J., Kolde, R., Berstler, J., Billings, K. J., Khor, B., Seashore-Ludlow, B., Fassl, A., Russell, C. N., Latorre, I. J., Jiang, B., Graham, D. B., Perez, J. R., Sicinski, P., Phillips, A. J., Schreiber, S. L., Gray, N. S., Shamji, A. F. & Xavier, R. J. Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells. Nat Chem Biol 13, 1102–1108 (2017). Johnson, P. R., Swanson, R., Rakhilina, L. & Hochstrasser, M. Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway. Cell 94, 217–227 (1998). Joos, B., Fischer, M., Kuster, H., Pillai, S. K., Wong, J. K., Böni, J., Hirschel, B., Weber, R., Trkola, A., Günthard, H. F., & Swiss HIV Cohort Study. HIV rebounds from latently infected cells, rather than from continuing low-level replication. Proc Natl Acad Sci U S A 105, 16725–16730 (2008). Jordan, A., Defechereux, P. & Verdin, E. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J 20, 1726–1738 (2001). 176 Jordan, A., Bisgrove, D. & Verdin, E. HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22, 1868–1877 (2003). Judge, M., Parker, E., Naniche, D. & Le Souëf, P. Gene Expression: the Key to Understanding HIV-1 Infection? Microbiol Mol Biol Rev 84, e00080-19 (2020). Julias, J. G., Ferris, A. L., Boyer, P. L. & Hughes, S. H. Replication of phenotypically mixed human immunodeficiency virus type 1 virions containing catalytically active and catalytically inactive reverse transcriptase. J Virol 75, 6537–6546 (2001). Julias, J. G., McWilliams, M. J., Sarafianos, S. G., Arnold, E. & Hughes, S. H. Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo. Proc Natl Acad Sci U S A 99, 9515–9520 (2002). Kanno, Y., Kure, Y., Kobayashi, S., Mizuno, M., Tsuchiya, Y., Yamashita, N., Nemoto, K. & Inouye, Y. Tripartite Motif Containing 24 Acts as a Novel Coactivator of the Constitutive Active/Androstane Receptor. Drug Metab Dispos 46, 46–52 (2018). Kao, S. Y., Calman, A. F., Luciw, P. A. & Peterlin, B. M. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330, 489–493 (1987). Karn, J. The molecular biology of HIV latency: breaking and restoring the Tat-dependent transcriptional circuit. Curr Opin HIV AIDS 6, 4–11 (2011). Karn, J. & Stoltzfus, C. M. Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harb Perspect Med 2, a006916 (2012). Kauder, S. E., Bosque, A., Lindqvist, A., Planelles, V. & Verdin, E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog 5, e1000495 (2009). Kedei, N., Lundberg, D. J., Toth, A., Welburn, P., Garfield, S. H. & Blumberg, P. M. Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res 64, 3243–3255 (2004). Kessing, C. F., Nixon, C. C., Li, C., Tsai, P., Takata, H., Mousseau, G., Ho, P. T., Honeycutt, J. B., Fallahi, M., Trautmann, L., Garcia, J. V. & Valente, S. T. In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a ‘Block-and-Lock’ Strategy for HIV-1 Treatment. Cell Rep 21, 600–611 (2017). Khan, S., Iqbal, M., Tariq, M., Baig, S. M. & Abbas, W. Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins. Clin Epigenetics 10, 14 (2018). Kikuchi, M., Okumura, F., Tsukiyama, T., Watanabe, M., Miyajima, N., Tanaka, J., Imamura, M. & Hatakeyama, S. TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells. Biochim Biophys Acta 1793, 1828–1836 (2009). 177 Kim, D. W. & Cochran, B. H. Extracellular signal-regulated kinase binds to TFII-I and regulates its activation of the c-fos promoter. Mol Cell Biol 20, 1140–1148 (2000). Kim, Y. K., Bourgeois, C. F., Isel, C., Churcher, M. J. & Karn, J. Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. Mol Cell Biol 22, 4622–4637 (2002). Kim, Y. K., Bourgeois, C. F., Pearson, R., Tyagi, M., West, M. J., Wong, J., Wu, S.-Y., Chiang, C.-M. & Karn, J. Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. EMBO J 25, 3596–3604 (2006). Kim, Y., Anderson, J. L. & Lewin, S. R. Getting the ‘Kill’ into ‘Shock and Kill’: Strategies to Eliminate Latent HIV. Cell Host Microbe 23, 14–26 (2018). Klaver, B. & Berkhout, B. Comparison of 5’ and 3’ long terminal repeat promoter function in human immunodeficiency virus. J Virol 68, 3830–3840 (1994). Komarnitsky, P., Cho, E. J. & Buratowski, S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev 14, 2452–2460 (2000). Korin, Y. D. & Zack, J. A. Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells. J Virol 72, 3161–3168 (1998). Krasnopolsky, S., Kuzmina, A. & Taube, R. Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency. PLoS Pathog 16, e1008834 (2020). Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J. & Hendrickson, W. A. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659 (1998). Lahaye, X., Satoh, T., Gentili, M., Cerboni, S., Conrad, C., Hurbain, I., El Marjou, A., Lacabaratz, C., Lelièvre, J.-D. & Manel, N. The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells. Immunity 39, 1132–1142 (2013). Laird, G. M., Bullen, C. K., Rosenbloom, D. I. S., Martin, A. R., Hill, A. L., Durand, C. M., Siliciano, J. D. & Siliciano, R. F. Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations. J Clin Invest 125, 1901–1912 (2015). Laskey, S. B. & Siliciano, R. F. A mechanistic theory to explain the efficacy of antiretroviral therapy. Nat Rev Microbiol 12, 772–780 (2014). 178 Laspia, M. F., Rice, A. P. & Mathews, M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell 59, 283–292 (1989). Lenasi, T., Contreras, X. & Peterlin, B. M. Transcriptional interference antagonizes proviral gene expression to promote HIV latency. Cell Host Microbe 4, 123–133 (2008). Lewinski, M. K., Bisgrove, D., Shinn, P., Chen, H., Hoffmann, C., Hannenhalli, S., Verdin, E., Berry, C. C., Ecker, J. R. & Bushman, F. D. Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J Virol 79, 6610–6619 (2005). Li, L., Li, H. S., Pauza, C. D., Bukrinsky, M. & Zhao, R. Y. Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions. Cell Res 15, 923–934 (2005). Li, Z., Guo, J., Wu, Y. & Zhou, Q. The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation. Nucleic Acids Res 41, 277–287 (2013). Liu, Q., Yin, X., Languino, L. R. & Altieri, D. C. Evaluation of drug combination effect using a Bliss independence dose-response surface model. Stat Biopharm Res 10, 112–122 (2018). Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001). Llano, M., Saenz, D. T., Meehan, A., Wongthida, P., Peretz, M., Walker, W. H., Teo, W. & Poeschla, E. M. An essential role for LEDGF/p75 in HIV integration. Science 314, 461–464 (2006). Lu, H. K., Gray, L. R., Wightman, F., Ellenberg, P., Khoury, G., Cheng, W.-J., Mota, T. M., Wesselingh, S., Gorry, P. R., Cameron, P. U., Churchill, M. J. & Lewin, S. R. Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy. PLoS One 9, e113341 (2014). Lu, P., Shen, Y., Yang, H., Wang, Y., Jiang, Z., Yang, X., Zhong, Y., Pan, H., Xu, J., Lu, H. & Zhu, H. BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency. Sci Rep 7, 16646 (2017). Lucic, B. & Lusic, M. Connecting HIV-1 integration and transcription: a step toward new treatments. FEBS Lett 590, 1927–1939 (2016). Luo, X. & Sawadogo, M. Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains. Mol Cell Biol 16, 1367–1375 (1996). Luo, Y., Hitz, B. C., Gabdank, I., Hilton, J. A., Kagda, M. S., Lam, B., Myers, Z., Sud, P., Jou, J., Lin, K., Baymuradov, U. K., Graham, K., Litton, C., Miyasato, S. R., Strattan, J. S., Jolanki, O., Lee, J.-W., Tanaka, F. Y., Adenekan, P., O’Neill, E. & Cherry, J. M. New developments on 179 the Encyclopedia of DNA Elements (ENCODE) data portal. Nucleic Acids Res 48, D882–D889 (2020). Lupp, S., Götz, C., Khadouma, S., Horbach, T., Dimova, E. Y., Bohrer, A.-M., Kietzmann, T. & Montenarh, M. The upstream stimulatory factor USF1 is regulated by protein kinase CK2 phosphorylation. Cell Signal 26, 2809–2817 (2014). Lv, D., Li, Y., Zhang, W., Alvarez, A. A., Song, L., Tang, J., Gao, W.-Q., Hu, B., Cheng, S.-Y. & Feng, H. TRIM24 is an oncogenic transcriptional co-activator of STAT3 in glioblastoma. Nat Commun 8, 1454 (2017). Ma, L., Yuan, L., An, J., Barton, M. C., Zhang, Q. & Liu, Z. Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer. Tumour Biol 37, 14803–14812 (2016). Ma, X., Yang, T., Luo, Y., Wu, L., Jiang, Y., Song, Z., Pan, T., Liu, B., Liu, G., Liu, J., Yu, F., He, Z., Zhang, W., Yang, J., Liang, L., Guan, Y., Zhang, X., Li, L., Cai, W., Tang, X., Gao, S., Deng, K. & Zhang, H. TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb. Elife 8, e42426 (2019). Maekawa, T., Sudo, T., Kurimoto, M. & Ishii, S. USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1. Nucleic Acids Res 19, 4689–4694 (1991). Malcolm, T., Chen, J., Chang, C. & Sadowski, I. Induction of chromosomally integrated HIV-1 LTR requires RBF-2 (USF/TFII-I) and Ras/MAPK signaling. Virus Genes 35, 215–223 (2007). Malcolm, T., Kam, J., Pour, P. S. & Sadowski, I. Specific interaction of TFII-I with an upstream element on the HIV-1 LTR regulates induction of latent provirus. FEBS Lett 582, 3903–3908 (2008). Maldarelli, F., Wu, X., Su, L., Simonetti, F. R., Shao, W., Hill, S., Spindler, J., Ferris, A. L., Mellors, J. W., Kearney, M. F., Coffin, J. M. & Hughes, S. H. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells. Science 345, 179–183 (2014). Mamede, J. I., Cianci, G. C., Anderson, M. R. & Hope, T. J. Early cytoplasmic uncoating is associated with infectivity of HIV-1. Proc Natl Acad Sci U S A 114, E7169–E7178 (2017). Marban, C., Redel, L., Suzanne, S., Van Lint, C., Lecestre, D., Chasserot-Golaz, S., Leid, M., Aunis, D., Schaeffer, E. & Rohr, O. COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells. Nucleic Acids Res 33, 2318–2331 (2005). Marban, C., Suzanne, S., Dequiedt, F., de Walque, S., Redel, L., Van Lint, C., Aunis, D. & Rohr, O. Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J 26, 412–423 (2007). 180 Margolis, D. M. & Archin, N. M. Proviral Latency, Persistent Human Immunodeficiency Virus Infection, and the Development of Latency Reversing Agents. J Infect Dis 215, S111–S118 (2017). Mattei, A. L., Bailly, N. & Meissner, A. DNA methylation: a historical perspective. Trends Genet 38, 676–707 (2022). Mbonye, U. & Karn, J. Transcriptional control of HIV latency: cellular signaling pathways, epigenetics, happenstance and the hope for a cure. Virology 454–455, 328–339 (2014). Mbonye, U. R., Gokulrangan, G., Datt, M., Dobrowolski, C., Cooper, M., Chance, M. R. & Karn, J. Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+) T lymphocytes. PLoS Pathog 9, e1003338 (2013). Mbonye, U., Wang, B., Gokulrangan, G., Shi, W., Yang, S. & Karn, J. Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation. J Biol Chem 293, 10009–10025 (2018). McAvera, R. M. & Crawford, L. J. TIF1 Proteins in Genome Stability and Cancer. Cancers (Basel) 12, 2094 (2020). McDonald, D., Vodicka, M. A., Lucero, G., Svitkina, T. M., Borisy, G. G., Emerman, M. & Hope, T. J. Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159, 441–452 (2002). McManus, W. R., Bale, M. J., Spindler, J., Wiegand, A., Musick, A., Patro, S. C., Sobolewski, M. D., Musick, V. K., Anderson, E. M., Cyktor, J. C., Halvas, E. K., Shao, W., Wells, D., Wu, X., Keele, B. F., Milush, J. M., Hoh, R., Mellors, J. W., Hughes, S. H., Deeks, S. G., Coffin, J. M. & Kearney, M. F. HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy. J Clin Invest 129, 4629–4642 (2019). McNamara, R. P., Reeder, J. E., McMillan, E. A., Bacon, C. W., McCann, J. L. & D’Orso, I. KAP1 Recruitment of the 7SK snRNP Complex to Promoters Enables Transcription Elongation by RNA Polymerase II. Mol Cell 61, 39–53 (2016). Mediouni, S., Chinthalapudi, K., Ekka, M. K., Usui, I., Jablonski, J. A., Clementz, M. A., Mousseau, G., Nowak, J., Macherla, V. R., Beverage, J. N., Esquenazi, E., Baran, P., de Vera, I. M. S., Kojetin, D., Loret, E. P., Nettles, K., Maiti, S., Izard, T. & Valente, S. T. Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat. mBio 10, e02662-18 (2019). Michael, N. L., Vahey, M. T., d’Arcy, L., Ehrenberg, P. K., Mosca, J. D., Rappaport, J. & Redfield, R. R. Negative-strand RNA transcripts are produced in human immunodeficiency virus type 1-infected cells and patients by a novel promoter downregulated by Tat. J Virol 68, 979–987 (1994). 181 Miyamoto, N. G., Moncollin, V., Egly, J. M. & Chambon, P. Specific interaction between a transcription factor and the upstream element of the adenovirus-2 major late promoter. EMBO J 4, 3563–3570 (1985). Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). Moore, M. D. & Hu, W. S. HIV-1 RNA dimerization: It takes two to tango. AIDS Rev 11, 91–102 (2009). Moranguinho, I. & Valente, S. T. Block-And-Lock: New Horizons for a Cure for HIV-1. Viruses 12, 1443 (2020). Mori, L., Jenike, K., Yeh, Y.-H. J., Lacombe, B., Li, C., Getzler, A., Mediouni, S., Cameron, M., Pipkin, M., Ho, Y.-C., Ramirez, B. C. & Valente, S. The XPB Subunit of the TFIIH Complex Plays a Critical Role in HIV-1 Transcription and XPB Inhibition by Spironolactone Prevents HIV-1 Reactivation from Latency. J Virol 95, e01247-20, JVI.01247-20 (2021). Morton, E. L., Forst, C. V., Zheng, Y., DePaula-Silva, A. B., Ramirez, N.-G. P., Planelles, V. & D’Orso, I. Transcriptional Circuit Fragility Influences HIV Proviral Fate. Cell Rep 27, 154-171.e9 (2019). Mousseau, G., Clementz, M. A., Bakeman, W. N., Nagarsheth, N., Cameron, M., Shi, J., Baran, P., Fromentin, R., Chomont, N. & Valente, S. T. An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription. Cell Host Microbe 12, 97–108 (2012). Mousseau, G., Kessing, C. F., Fromentin, R., Trautmann, L., Chomont, N. & Valente, S. T. The Tat Inhibitor Didehydro-Cortistatin A Prevents HIV-1 Reactivation from Latency. mBio 6, e00465 (2015). Muesing, M. A., Smith, D. H. & Capon, D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell 48, 691–701 (1987). Müller, T. G., Zila, V., Müller, B. & Kräusslich, H.-G. Nuclear Capsid Uncoating and Reverse Transcription of HIV-1. Annu Rev Virol 9, 261–284 (2022). Naghavi, M. H., Estable, M. C., Schwartz, S., Roeder, R. G. & Vahlne, A. Upstream stimulating factor affects human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in a cell-specific manner, independently of the HIV-1 subtype and the core-negative regulatory element. J Gen Virol 82, 547–559 (2001). 182 Nemet, J., Jelicic, B., Rubelj, I. & Sopta, M. The two faces of Cdk8, a positive/negative regulator of transcription. Biochimie 97, 22–27 (2014). Nguyen, K., Das, B., Dobrowolski, C. & Karn, J. Multiple Histone Lysine Methyltransferases Are Required for the Establishment and Maintenance of HIV-1 Latency. mBio 8, e00133-17 (2017). Nowak, D. E., Tian, B., Jamaluddin, M., Boldogh, I., Vergara, L. A., Choudhary, S. & Brasier, A. R. RelA Ser276 phosphorylation is required for activation of a subset of NF-kappaB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes. Mol Cell Biol 28, 3623–3638 (2008). Nowak, M., Helleboid-Chapman, A., Jakel, H., Martin, G., Duran-Sandoval, D., Staels, B., Rubin, E. M., Pennacchio, L. A., Taskinen, M.-R., Fruchart-Najib, J. & Fruchart, J.-C. Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor. Mol Cell Biol 25, 1537–1548 (2005). Nyamweya, S., Hegedus, A., Jaye, A., Rowland-Jones, S., Flanagan, K. L. & Macallan, D. C. Comparing HIV-1 and HIV-2 infection: Lessons for viral immunopathogenesis. Rev Med Virol 23, 221–240 (2013). Olave, N. C., Grenett, M. H., Cadeiras, M., Grenett, H. E. & Higgins, P. J. Upstream stimulatory factor-2 mediates quercetin-induced suppression of PAI-1 gene expression in human endothelial cells. J Cell Biochem 111, 720–726 (2010). Osman, S., Mohammad, E., Lidschreiber, M., Stuetzer, A., Bazsó, F. L., Maier, K. C., Urlaub, H. & Cramer, P. The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II. J Biol Chem 296, 100734 (2021). Ott, M., Geyer, M. & Zhou, Q. The control of HIV transcription: keeping RNA polymerase II on track. Cell Host Microbe 10, 426–435 (2011). Palacios, J. A., Pérez-Piñar, T., Toro, C., Sanz-Minguela, B., Moreno, V., Valencia, E., Gómez-Hernando, C. & Rodés, B. Long-term nonprogressor and elite controller patients who control viremia have a higher percentage of methylation in their HIV-1 proviral promoters than aviremic patients receiving highly active antiretroviral therapy. J Virol 86, 13081–13084 (2012). Palella, F. J., Delaney, K. M., Moorman, A. C., Loveless, M. O., Fuhrer, J., Satten, G. A., Aschman, D. J. & Holmberg, S. D. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 338, 853–860 (1998). Palmer, W. S., Poncet-Montange, G., Liu, G., Petrocchi, A., Reyna, N., Subramanian, G., Theroff, J., Yau, A., Kost-Alimova, M., Bardenhagen, J. P., Leo, E., Shepard, H. E., Tieu, T. N., 183 Shi, X., Zhan, Y., Zhao, S., Barton, M. C., Draetta, G., Toniatti, C., Jones, P., Geck Do, M. & Andersen, J. N. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J Med Chem 59, 1440–1454 (2016). Panganiban, A. T. & Fiore, D. Ordered interstrand and intrastrand DNA transfer during reverse transcription. Science 241, 1064–1069 (1988). Pasquereau, S. & Herbein, G. CounterAKTing HIV: Toward a ‘Block and Clear’ Strategy? Front Cell Infect Microbiol 12, 827717 (2022). Pathiraja, T. N., Thakkar, K. N., Jiang, S., Stratton, S., Liu, Z., Gagea, M., Shi, X., Shah, P. K., Phan, L., Lee, M.-H., Andersen, J., Stampfer, M. & Barton, M. C. TRIM24 links glucose metabolism with transformation of human mammary epithelial cells. Oncogene 34, 2836–2845 (2015). Pau, A. K. & George, J. M. Antiretroviral therapy: current drugs. Infect Dis Clin North Am 28, 371–402 (2014). Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadonaga, J. T. & Jones, K. A. NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro. Genes Dev 10, 37–49 (1996). Pearson, R., Kim, Y. K., Hokello, J., Lassen, K., Friedman, J., Tyagi, M. & Karn, J. Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol 82, 12291–12303 (2008). Pelish, H. E., Liau, B. B., Nitulescu, I. I., Tangpeerachaikul, A., Poss, Z. C., Da Silva, D. H., Caruso, B. T., Arefolov, A., Fadeyi, O., Christie, A. L., Du, K., Banka, D., Schneider, E. V., Jestel, A., Zou, G., Si, C., Ebmeier, C. C., Bronson, R. T., Krivtsov, A. V., Myers, A. G., Kohl, N. E., Kung, A. L., Armstrong, S. A., Lemieux, M. E., Taatjes, D. J. & Shair, M. D. Mediator kinase inhibition further activates super-enhancer-associated genes in AML. Nature 526, 273–276 (2015). Peng, H., Begg, G. E., Schultz, D. C., Friedman, J. R., Jensen, D. E., Speicher, D. W. & Rauscher, F. J. Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions. J Mol Biol 295, 1139–1162 (2000). Pereira, L. A., Bentley, K., Peeters, A., Churchill, M. J. & Deacon, N. J. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res 28, 663–668 (2000). 184 Perez-Lloret, J., Okoye, I. S., Guidi, R., Kannan, Y., Coomes, S. M., Czieso, S., Mengus, G., Davidson, I. & Wilson, M. S. T-cell-intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy. Proc Natl Acad Sci U S A 113, E568-576 (2016). Peruski, A. H., Wesolowski, L. G., Delaney, K. P., Chavez, P. R., Owen, S. M., Granade, T. C., Sullivan, V., Switzer, W. M., Dong, X., Brooks, J. T. & Joyce, M. P. Trends in HIV-2 Diagnoses and Use of the HIV-1/HIV-2 Differentiation Test - United States, 2010-2017. MMWR Morb Mortal Wkly Rep 69, 63–66 (2020). Piller, S. C., Caly, L. & Jans, D. A. Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV? Curr Drug Targets 4, 409–429 (2003). Ping, Y. H. & Rana, T. M. DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation. J Biol Chem 276, 12951–12958 (2001). Plesa, G., Dai, J., Baytop, C., Riley, J. L., June, C. H. & O’Doherty, U. Addition of deoxynucleosides enhances human immunodeficiency virus type 1 integration and 2LTR formation in resting CD4+ T cells. J Virol 81, 13938–13942 (2007). Porter, D. C., Farmaki, E., Altilia, S., Schools, G. P., West, D. K., Chen, M., Chang, B.-D., Puzyrev, A. T., Lim, C., Rokow-Kittell, R., Friedhoff, L. T., Papavassiliou, A. G., Kalurupalle, S., Hurteau, G., Shi, J., Baran, P. S., Gyorffy, B., Wentland, M. P., Broude, E. V., Kiaris, H. & Roninson, I. B. Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities. Proc Natl Acad Sci U S A 109, 13799–13804 (2012). Qi, L., Higgins, C. E., Higgins, S. P., Law, B. K., Simone, T. M. & Higgins, P. J. The basic helix-loop-helix/leucine zipper transcription factor USF2 integrates serum-induced PAI-1 expression and keratinocyte growth. J Cell Biochem 115, 1840–1847 (2014). Qu, D., Sun, W.-W., Li, L., Ma, L., Sun, L., Jin, X., Li, T., Hou, W. & Wang, J.-H. Long noncoding RNA MALAT1 releases epigenetic silencing of HIV-1 replication by displacing the polycomb repressive complex 2 from binding to the LTR promoter. Nucleic Acids Res 47, 3013–3027 (2019). Qyang, Y., Luo, X., Lu, T., Ismail, P. M., Krylov, D., Vinson, C. & Sawadogo, M. Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation. Mol Cell Biol 19, 1508–1517 (1999). Rada-Iglesias, A., Ameur, A., Kapranov, P., Enroth, S., Komorowski, J., Gingeras, T. R. & Wadelius, C. Whole-genome maps of USF1 and USF2 binding and histone H3 acetylation reveal new aspects of promoter structure and candidate genes for common human disorders. Genome Res 18, 380–392 (2008). 185 Rafati, H., Parra, M., Hakre, S., Moshkin, Y., Verdin, E. & Mahmoudi, T. Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency. PLoS Biol 9, e1001206 (2011). Rando, O. J. Global patterns of histone modifications. Curr Opin Genet Dev 17, 94–99 (2007). Rankovic, S., Varadarajan, J., Ramalho, R., Aiken, C. & Rousso, I. Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly. J Virol 91, e00289-17 (2017). Rasaiyaah, J., Tan, C. P., Fletcher, A. J., Price, A. J., Blondeau, C., Hilditch, L., Jacques, D. A., Selwood, D. L., James, L. C., Noursadeghi, M. & Towers, G. J. HIV-1 evades innate immune recognition through specific cofactor recruitment. Nature 503, 402–405 (2013). Rasmussen, T. A., Tolstrup, M., Brinkmann, C. R., Olesen, R., Erikstrup, C., Solomon, A., Winckelmann, A., Palmer, S., Dinarello, C., Buzon, M., Lichterfeld, M., Lewin, S. R., Østergaard, L. & Søgaard, O. S. Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV 1, e13-21 (2014). Razooky, B. S., Pai, A., Aull, K., Rouzine, I. M. & Weinberger, L. S. A hardwired HIV latency program. Cell 160, 990–1001 (2015). Razooky, B. S. & Weinberger, L. S. Mapping the architecture of the HIV-1 Tat circuit: A decision-making circuit that lacks bistability and exploits stochastic noise. Methods 53, 68–77 (2011). Read, D. F., Atindaana, E., Pyaram, K., Yang, F., Emery, S., Cheong, A., Nakama, K. R., Burnett, C., Larragoite, E. T., Battivelli, E., Verdin, E., Planelles, V., Chang, C.-H., Telesnitsky, A. & Kidd, J. M. Stable integrant-specific differences in bimodal HIV-1 expression patterns revealed by high-throughput analysis. PLoS Pathog 15, e1007903 (2019). Regoes, R. R. & Bonhoeffer, S. The HIV coreceptor switch: a population dynamical perspective. Trends Microbiol 13, 269–277 (2005). Rein, A. RNA Packaging in HIV. Trends Microbiol 27, 715–723 (2019). Reuse, S., Calao, M., Kabeya, K., Guiguen, A., Gatot, J.-S., Quivy, V., Vanhulle, C., Lamine, A., Vaira, D., Demonte, D., Martinelli, V., Veithen, E., Cherrier, T., Avettand, V., Poutrel, S., Piette, J., de Launoit, Y., Moutschen, M., Burny, A., Rouzioux, C., De Wit, S., Herbein, G., Rohr, O., Collette, Y., Lambotte, O., Clumeck, N. & Van Lint, C. Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection. PLoS One 4, e6093 (2009). Richmond, T. J. & Davey, C. A. The structure of DNA in the nucleosome core. Nature 423, 145–150 (2003). 186 Rizzuto, C. D., Wyatt, R., Hernández-Ramos, N., Sun, Y., Kwong, P. D., Hendrickson, W. A. & Sodroski, J. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 280, 1949–1953 (1998). Roy, A. L., Du, H., Gregor, P. D., Novina, C. D., Martinez, E. & Roeder, R. G. Cloning of an inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1. EMBO J 16, 7091–7104 (1997). Roy, A. L., Meisterernst, M., Pognonec, P. & Roeder, R. G. Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF. Nature 354, 245–248 (1991). Roy, A. L. Biochemistry and biology of the inducible multifunctional transcription factor TFII-I: 10 years later. Gene 492, 32–41 (2012). Roy, S., Sen, C. K., Kobuchi, H. & Packer, L. Antioxidant regulation of phorbol ester-induced adhesion of human Jurkat T-cells to endothelial cells. Free Radic Biol Med 25, 229–241 (1998). Sadowski, I. & Hashemi, F. B. Strategies to eradicate HIV from infected patients: elimination of latent provirus reservoirs. Cell Mol Life Sci 76, 3583–3600 (2019). Sadowski, I., Lourenco, P. & Malcolm, T. Factors controlling chromatin organization and nucleosome positioning for establishment and maintenance of HIV latency. Curr HIV Res 6, 286–295 (2008). Sadowski, I. & Mitchell, D. A. TFII-I and USF (RBF-2) regulate Ras/MAPK-responsive HIV-1 transcription in T cells. Eur J Cancer 41, 2528–2536 (2005). Saito, A. & Yamashita, M. HIV-1 capsid variability: viral exploitation and evasion of capsid-binding molecules. Retrovirology 18, 32 (2021). Sawadogo, M. & Roeder, R. G. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43, 165–175 (1985). Sawadogo, M., Van Dyke, M. W., Gregor, P. D. & Roeder, R. G. Multiple forms of the human gene-specific transcription factor USF. I. Complete purification and identification of USF from HeLa cell nuclei. J Biol Chem 263, 11985–11993 (1988). Sayasith, K., Lussier, J. G. & Sirois, J. Role of upstream stimulatory factor phosphorylation in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells. J Biol Chem 280, 28885–28893 (2005). Schröder, A. R. W., Shinn, P., Chen, H., Berry, C., Ecker, J. R. & Bushman, F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002). 187 Selby, M. J., Bain, E. S., Luciw, P. A. & Peterlin, B. M. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev 3, 547–558 (1989). Segura-Puimedon, M., Borralleras, C., Pérez-Jurado, L. A. & Campuzano, V. TFII-I regulates target genes in the PI-3K and TGF-β signaling pathways through a novel DNA binding motif. Gene 527, 529–536 (2013). Shah, V. B. & Aiken, C. In vitro uncoating of HIV-1 cores. J Vis Exp 3384 (2011). doi:10.3791/3384 Shah, V. V., Duncan, A. D., Jiang, S., Stratton, S. A., Allton, K. L., Yam, C., Jain, A., Krause, P. M., Lu, Y., Cai, S., Tu, Y., Zhou, X., Zhang, X., Jiang, Y., Carroll, C. L., Kang, Z., Liu, B., Shen, J., Gagea, M., Manu, S. M., Huo, L., Gilcrease, M., Powell, R. T., Guo, L., Stephan, C., Davies, P. J., Parker-Thornburg, J., Lozano, G., Behringer, R. R., Piwnica-Worms, H., Chang, J. T., Moulder, S. L. & Barton, M. C. Mammary-specific expression of Trim24 establishes a mouse model of human metaplastic breast cancer. Nat Commun 12, 5389 (2021). Shan, L. & Siliciano, R. F. From reactivation of latent HIV-1 to elimination of the latent reservoir: the presence of multiple barriers to viral eradication. Bioessays 35, 544–552 (2013). Shan, L., Yang, H.-C., Rabi, S. A., Bravo, H. C., Shroff, N. S., Irizarry, R. A., Zhang, H., Margolick, J. B., Siliciano, J. D. & Siliciano, R. F. Influence of host gene transcription level and orientation on HIV-1 latency in a primary-cell model. J Virol 85, 5384–5393 (2011). Shi, J., Manolikakes, G., Yeh, C.-H., Guerrero, C. A., Shenvi, R. A., Shigehisa, H. & Baran, P. S. Scalable synthesis of cortistatin A and related structures. J Am Chem Soc 133, 8014–8027 (2011). Shi, J., Shigehisa, H., Guerrero, C. A., Shenvi, R. A., Li, C.-C. & Baran, P. S. Stereodivergent synthesis of 17-alpha and 17-beta-alpharyl steroids: application and biological evaluation of D-ring cortistatin analogues. Angew Chem Int Ed Engl 48, 4328–4331 (2009). Shieh, B. H., Sparkes, R. S., Gaynor, R. B. & Lusis, A. J. Localization of the gene-encoding upstream stimulatory factor (USF) to human chromosome 1q22-q23. Genomics 16, 266–268 (1993). Shimabukuro-Vornhagen, A., Gödel, P., Subklewe, M., Stemmler, H. J., Schlößer, H. A., Schlaak, M., Kochanek, M., Böll, B. & von Bergwelt-Baildon, M. S. Cytokine release syndrome. J Immunother Cancer 6, 56 (2018). Shimizu, N., Haraguchi, Y., Takeuchi, Y., Soda, Y., Kanbe, K. & Hoshino, H. Changes in and discrepancies between cell tropisms and coreceptor uses of human immunodeficiency virus type 1 induced by single point mutations at the V3 tip of the env protein. Virology 259, 324–333 (1999). 188 Shun, M.-C., Raghavendra, N. K., Vandegraaff, N., Daigle, J. E., Hughes, S., Kellam, P., Cherepanov, P. & Engelman, A. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev 21, 1767–1778 (2007). Sieweke, M. H., Tekotte, H., Jarosch, U. & Graf, T. Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells. EMBO J 17, 1728–1739 (1998). Siliciano, R. F. & Greene, W. C. HIV latency. Cold Spring Harb Perspect Med 1, a007096 (2011). Simonetti, F. R., Zhang, H., Soroosh, G. P., Duan, J., Rhodehouse, K., Hill, A. L., Beg, S. A., McCormick, K., Raymond, H. E., Nobles, C. L., Everett, J. K., Kwon, K. J., White, J. A., Lai, J., Margolick, J. B., Hoh, R., Deeks, S. G., Bushman, F. D., Siliciano, J. D. & Siliciano, R. F. Antigen-driven clonal selection shapes the persistence of HIV-1-infected CD4+ T cells in vivo. J Clin Invest 131, e145254, 145254 (2021). Singh, A., Razooky, B., Cox, C. D., Simpson, M. L. & Weinberger, L. S. Transcriptional bursting from the HIV-1 promoter is a significant source of stochastic noise in HIV-1 gene expression. Biophys J 98, L32-34 (2010). Singh, I., Ozturk, N., Cordero, J., Mehta, A., Hasan, D., Cosentino, C., Sebastian, C., Krüger, M., Looso, M., Carraro, G., Bellusci, S., Seeger, W., Braun, T., Mostoslavsky, R. & Barreto, G. High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX. Cell Res 25, 837–850 (2015). Sirito, M., Lin, Q., Deng, J. M., Behringer, R. R. & Sawadogo, M. Overlapping roles and asymmetrical cross-regulation of the USF proteins in mice. Proc Natl Acad Sci U S A 95, 3758–3763 (1998). Sirito, M., Lin, Q., Maity, T. & Sawadogo, M. Ubiquitous expression of the 43- and 44-kDa forms of transcription factor USF in mammalian cells. Nucleic Acids Res 22, 427–433 (1994). Sirito, M., Walker, S., Lin, Q., Kozlowski, M. T., Klein, W. H. & Sawadogo, M. Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers. Gene Expr 2, 231–240 (1992). Sloan, R. D. & Wainberg, M. A. The role of unintegrated DNA in HIV infection. Retrovirology 8, 52 (2011). Smith, S. J., Zhao, X. Z., Passos, D. O., Lyumkis, D., Burke, T. R. & Hughes, S. H. Integrase Strand Transfer Inhibitors Are Effective Anti-HIV Drugs. Viruses 13, 205 (2021). Sodroski, J., Patarca, R., Rosen, C., Wong-Staal, F. & Haseltine, W. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science 229, 74–77 (1985). 189 Sodroski, J., Rosen, C., Wong-Staal, F., Salahuddin, S. Z., Popovic, M., Arya, S., Gallo, R. C. & Haseltine, W. A. Trans-acting transcriptional regulation of human T-cell leukemia virus type III long terminal repeat. Science 227, 171–173 (1985). Søgaard, O. S., Graversen, M. E., Leth, S., Olesen, R., Brinkmann, C. R., Nissen, S. K., Kjaer, A. S., Schleimann, M. H., Denton, P. W., Hey-Cunningham, W. J., Koelsch, K. K., Pantaleo, G., Krogsgaard, K., Sommerfelt, M., Fromentin, R., Chomont, N., Rasmussen, T. A., Østergaard, L. & Tolstrup, M. The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo. PLoS Pathog 11, e1005142 (2015). Spohrer, S., Groß, R., Nalbach, L., Schwind, L., Stumpf, H., Menger, M. D., Ampofo, E., Montenarh, M. & Götz, C. Functional interplay between the transcription factors USF1 and PDX-1 and protein kinase CK2 in pancreatic β-cells. Sci Rep 7, 16367 (2017). Stevenson, M., Stanwick, T. L., Dempsey, M. P. & Lamonica, C. A. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J 9, 1551–1560 (1990). Struhl, K. & Segal, E. Determinants of nucleosome positioning. Nat Struct Mol Biol 20, 267–273 (2013). Sumner, R. P., Harrison, L., Touizer, E., Peacock, T. P., Spencer, M., Zuliani-Alvarez, L. & Towers, G. J. Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA. EMBO J 39, e103958 (2020). Swiggard, W. J., Baytop, C., Yu, J. J., Dai, J., Li, C., Schretzenmair, R., Theodosopoulos, T. & O’Doherty, U. Human immunodeficiency virus type 1 can establish latent infection in resting CD4+ T cells in the absence of activating stimuli. J Virol 79, 14179–14188 (2005). Tahirov, T. H., Babayeva, N. D., Varzavand, K., Cooper, J. J., Sedore, S. C. & Price, D. H. Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature 465, 747–751 (2010). Takizawa, T., Meaburn, K. J. & Misteli, T. The meaning of gene positioning. Cell 135, 9–13 (2008). Tan, Q., Zhu, Y., Li, J., Chen, Z., Han, G. W., Kufareva, I., Li, T., Ma, L., Fenalti, G., Li, J., Zhang, W., Xie, X., Yang, H., Jiang, H., Cherezov, V., Liu, H., Stevens, R. C., Zhao, Q. & Wu, B. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013). Taura, M., Song, E., Ho, Y.-C. & Iwasaki, A. Apobec3A maintains HIV-1 latency through recruitment of epigenetic silencing machinery to the long terminal repeat. Proc Natl Acad Sci U S A 116, 2282–2289 (2019). Terragni, J., Nayak, G., Banerjee, S., Medrano, J.-L., Graham, J. R., Brennan, J. F., Sepulveda, S. & Cooper, G. M. The E-box binding factors Max/Mnt, MITF, and USF1 act coordinately with 190 FoxO to regulate expression of proapoptotic and cell cycle control genes by phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 signaling. J Biol Chem 286, 36215–36227 (2011). The Committee for Drug Evaluation and Therapy,. Therapeutic Guidelines for Antiretroviral (ARV) Treatment of Adult HIV Infection. (2023). at <https://bccfe.ca/therapeutic-guidelines/guidelines-antiretroviral-arv-treatment-adult-hiv-infection> The Lancet, null. 40 years of HIV/AIDS: a painful anniversary. Lancet 397, 2125 (2021). Thénot, S., Henriquet, C., Rochefort, H. & Cavaillès, V. Differential interaction of nuclear receptors with the putative human transcriptional coactivator hTIF1. J Biol Chem 272, 12062–12068 (1997). Thorlund, K., Horwitz, M. S., Fife, B. T., Lester, R. & Cameron, D. W. Landscape review of current HIV ‘kick and kill’ cure research - some kicking, not enough killing. BMC Infect Dis 17, 595 (2017). Tiruppathi, C., Soni, D., Wang, D.-M., Xue, J., Singh, V., Thippegowda, P. B., Cheppudira, B. P., Mishra, R. K., Debroy, A., Qian, Z., Bachmaier, K., Zhao, Y.-Y., Christman, J. W., Vogel, S. M., Ma, A. & Malik, A. B. The transcription factor DREAM represses the deubiquitinase A20 and mediates inflammation. Nat Immunol 15, 239–247 (2014). Tisserand, J., Khetchoumian, K., Thibault, C., Dembélé, D., Chambon, P. & Losson, R. Tripartite motif 24 (Trim24/Tif1α) tumor suppressor protein is a novel negative regulator of interferon (IFN)/signal transducers and activators of transcription (STAT) signaling pathway acting through retinoic acid receptor α (Rarα) inhibition. J Biol Chem 286, 33369–33379 (2011). Tripathy, M. K., McManamy, M. E. M., Burch, B. D., Archin, N. M. & Margolis, D. M. H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4+ T Cells. J Virol 89, 8392–8405 (2015). Trujillo, J. R., Wang, W. K., Lee, T. H. & Essex, M. Identification of the envelope V3 loop as a determinant of a CD4-negative neuronal cell tropism for HIV-1. Virology 217, 613–617 (1996). Tsai, W.-W., Wang, Z., Yiu, T. T., Akdemir, K. C., Xia, W., Winter, S., Tsai, C.-Y., Shi, X., Schwarzer, D., Plunkett, W., Aronow, B., Gozani, O., Fischle, W., Hung, M.-C., Patel, D. J. & Barton, M. C. TRIM24 links a non-canonical histone signature to breast cancer. Nature 468, 927–932 (2010). Tseng, A., Seet, J. & Phillips, E. J. The evolution of three decades of antiretroviral therapy: challenges, triumphs and the promise of the future. Br J Clin Pharmacol 79, 182–194 (2015). Tyagi, M. & Karn, J. CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J 26, 4985–4995 (2007). 191 Tyagi, M., Pearson, R. J. & Karn, J. Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 84, 6425–6437 (2010). UNAIDS. AIDS epidemic update: December 2004. Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO). (2004). UNAIDS. IN DANGER: UNAIDS Global AIDS Update 2022. Geneva: Joint United Nations Programme on HIV/ AIDS. (2022). UNAIDS. Global HIV & AIDS statistics — Fact sheet. (2023). Unutmaz, D., KewalRamani, V. N., Marmon, S. & Littman, D. R. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J Exp Med 189, 1735–1746 (1999). Valente, S. T., Gilmartin, G. M., Venkatarama, K., Arriagada, G. & Goff, S. P. HIV-1 mRNA 3’ end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8. Mol Cell 36, 279–289 (2009). van Wamel, J. L. & Berkhout, B. The first strand transfer during HIV-1 reverse transcription can occur either intramolecularly or intermolecularly. Virology 244, 245–251 (1998). Vansant, G., Bruggemans, A., Janssens, J. & Debyser, Z. Block-And-Lock Strategies to Cure HIV Infection. Viruses 12, 84 (2020). Verdin, E. DNase I-hypersensitive sites are associated with both long terminal repeats and with the intragenic enhancer of integrated human immunodeficiency virus type 1. J Virol 65, 6790–6799 (1991). Verdin, E., Paras, P. & Van Lint, C. Chromatin disruption in the promoter of human immunodeficiency virus type 1 during transcriptional activation. EMBO J 12, 3249–3259 (1993). Viollet, B., Lefrançois-Martinez, A. M., Henrion, A., Kahn, A., Raymondjean, M. & Martinez, A. Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms. J Biol Chem 271, 1405–1415 (1996). Vullhorst, D. & Buonanno, A. Multiple GTF2I-like repeats of general transcription factor 3 exhibit DNA binding properties. Evidence for a common origin as a sequence-specific DNA interaction module. J Biol Chem 280, 31722–31731 (2005). Wagner, T. A., McKernan, J. L., Tobin, N. H., Tapia, K. A., Mullins, J. I. & Frenkel, L. M. An increasing proportion of monotypic HIV-1 DNA sequences during antiretroviral treatment suggests proliferation of HIV-infected cells. J Virol 87, 1770–1778 (2013). Wagner, T. A., McLaughlin, S., Garg, K., Cheung, C. Y. K., Larsen, B. B., Styrchak, S., Huang, H. C., Edlefsen, P. T., Mullins, J. I. & Frenkel, L. M. HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection. Science 345, 570–573 (2014). 192 Walker-Sperling, V. E., Pohlmeyer, C. W., Veenhuis, R. T., May, M., Luna, K. A., Kirkpatrick, A. R., Laeyendecker, O., Cox, A. L., Carrington, M., Bailey, J. R., Arduino, R. C. & Blankson, J. N. Factors Associated With the Control of Viral Replication and Virologic Breakthrough in a Recently Infected HIV-1 Controller. EBioMedicine 16, 141–149 (2017). Wang, G. Z., Wang, Y. & Goff, S. P. Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry. Cell Host Microbe 20, 798–809 (2016). Wang, Y., Wong, R. H. F., Tang, T., Hudak, C. S., Yang, D., Duncan, R. E. & Sul, H. S. Phosphorylation and recruitment of BAF60c in chromatin remodeling for lipogenesis in response to insulin. Mol Cell 49, 283–297 (2013). Wei, P., Garber, M. E., Fang, S. M., Fischer, W. H. & Jones, K. A. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92, 451–462 (1998). Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag, M. S., Wu, X., Shaw, G. M. & Kappes, J. C. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46, 1896–1905 (2002). Weinberger, L. S., Burnett, J. C., Toettcher, J. E., Arkin, A. P. & Schaffer, D. V. Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity. Cell 122, 169–182 (2005). Weinberger, L. S. & Shenk, T. An HIV feedback resistor: auto-regulatory circuit deactivator and noise buffer. PLoS Biol 5, e9 (2007). Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J. & Wiley, D. C. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387, 426–430 (1997). Welker, R., Hohenberg, H., Tessmer, U., Huckhagel, C. & Kräusslich, H. G. Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1. J Virol 74, 1168–1177 (2000). Wen, Y.-D., Cress, W. D., Roy, A. L. & Seto, E. Histone deacetylase 3 binds to and regulates the multifunctional transcription factor TFII-I. J Biol Chem 278, 1841–1847 (2003). West, A. G., Huang, S., Gaszner, M., Litt, M. D. & Felsenfeld, G. Recruitment of histone modifications by USF proteins at a vertebrate barrier element. Mol Cell 16, 453–463 (2004). Widlak, P., Gaynor, R. B. & Garrard, W. T. In vitro chromatin assembly of the HIV-1 promoter. ATP-dependent polar repositioning of nucleosomes by Sp1 and NFkappaB. J Biol Chem 272, 17654–17661 (1997). 193 Wienerroither, S., Shukla, P., Farlik, M., Majoros, A., Stych, B., Vogl, C., Cheon, H., Stark, G. R., Strobl, B., Müller, M. & Decker, T. Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex. Cell Rep 12, 300–312 (2015). Wilbourne, M. & Zhang, P. Visualizing HIV-1 Capsid and Its Interactions with Antivirals and Host Factors. Viruses 13, 246 (2021). Williams, S. A., Chen, L.-F., Kwon, H., Ruiz-Jarabo, C. M., Verdin, E. & Greene, W. C. NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 25, 139–149 (2006). Woldemeskel, B. A., Kwaa, A. K. & Blankson, J. N. Viral reservoirs in elite controllers of HIV-1 infection: Implications for HIV cure strategies. EBioMedicine 62, 103118 (2020). Wong, J. K., Hezareh, M., Günthard, H. F., Havlir, D. V., Ignacio, C. C., Spina, C. A. & Richman, D. D. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278, 1291–1295 (1997). Wong, R. H. F., Chang, I., Hudak, C. S. S., Hyun, S., Kwan, H.-Y. & Sul, H. S. A role of DNA-PK for the metabolic gene regulation in response to insulin. Cell 136, 1056–1072 (2009). Wong, R. H. F. & Sul, H. S. DNA-PK: relaying the insulin signal to USF in lipogenesis. Cell Cycle 8, 1977–1978 (2009). Wu, D., Zhang, Z., Chen, X., Yan, Y. & Liu, X. Angel or Devil ? - CDK8 as the new drug target. Eur J Med Chem 213, 113043 (2021). Xiao, Q., Kenessey, A. & Ojamaa, K. Role of USF1 phosphorylation on cardiac alpha-myosin heavy chain promoter activity. Am J Physiol Heart Circ Physiol 283, H213-219 (2002). Yamada, T., Yamaguchi, Y., Inukai, N., Okamoto, S., Mura, T. & Handa, H. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol Cell 21, 227–237 (2006). Yamashita, M. & Engelman, A. N. Capsid-Dependent Host Factors in HIV-1 Infection. Trends Microbiol 25, 741–755 (2017). Yang, Y., Fricke, T. & Diaz-Griffero, F. Inhibition of reverse transcriptase activity increases stability of the HIV-1 core. J Virol 87, 683–687 (2013). Ylisastigui, L., Archin, N. M., Lehrman, G., Bosch, R. J. & Margolis, D. M. Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression. AIDS 18, 1101–1108 (2004). Yu, H., Jetzt, A. E., Ron, Y., Preston, B. D. & Dougherty, J. P. The nature of human immunodeficiency virus type 1 strand transfers. J Biol Chem 273, 28384–28391 (1998). 194 Yu, M., Teo, T., Yang, Y., Li, M., Long, Y., Philip, S., Noll, B., Heinemann, G. K., Diab, S., Eldi, P., Mekonnen, L., Anshabo, A. T., Rahaman, M. H., Milne, R., Hayball, J. D. & Wang, S. Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation. Eur J Med Chem 214, 113248 (2021). Yu, M., Teo, T., Yang, Y., Li, M., Long, Y., Philip, S., Noll, B., Heinemann, G. K., Diab, S., Eldi, P., Mekonnen, L., Anshabo, A. T., Rahaman, M. H., Milne, R., Hayball, J. D. & Wang, S. Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation. Eur J Med Chem 214, 113248 (2021). Yu, T., Gan, S., Zhu, Q., Dai, D., Li, N., Wang, H., Chen, X., Hou, D., Wang, Y., Pan, Q., Xu, J., Zhang, X., Liu, J., Pei, S., Peng, C., Wu, P., Romano, S., Mao, C., Huang, M., Zhu, X., Shen, K., Qin, J. & Xiao, Y. Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24. Nat Commun 10, 4353 (2019). Yukl, S. A., Kaiser, P., Kim, P., Telwatte, S., Joshi, S. K., Vu, M., Lampiris, H. & Wong, J. K. HIV latency in isolated patient CD4+ T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing. Sci Transl Med 10, eaap9927 (2018). Zack, J. A., Haislip, A. M., Krogstad, P. & Chen, I. S. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J Virol 66, 1717–1725 (1992). Zhan, Y., Kost-Alimova, M., Shi, X., Leo, E., Bardenhagen, J. P., Shepard, H. E., Appikonda, S., Vangamudi, B., Zhao, S., Tieu, T. N., Jiang, S., Heffernan, T. P., Marszalek, J. R., Toniatti, C., Draetta, G., Tyler, J., Barton, M., Jones, P., Palmer, W. S., Geck Do, M. K. & Andersen, J. N. Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery. Epigenetics Chromatin 8, 37 (2015). Zhang, H., Zhang, Y., Zhou, X., Wright, S., Hyle, J., Zhao, L., An, J., Zhao, X., Shao, Y., Xu, B., Lee, H.-M., Chen, T., Zhou, Y., Chen, X., Lu, R. & Li, C. Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen. Elife 9, e57858 (2020). Zhang, H., Jing, L., Liu, M., Goto, M., Lai, F., Liu, X., Sheng, L., Yang, Y., Yang, Y., Li, Y., Chen, X., Lee, K.-H. & Xiao, Z. Identification of 3, 4-disubstituted pyridine derivatives as novel CDK8 inhibitors. Eur J Med Chem 223, 113634 (2021). Zhang, L., Cheng, C., Li, J., Wang, L., Chumanevich, A. A., Porter, D. C., Mindich, A., Gorbunova, S., Roninson, I. B., Chen, M. & McInnes, C. A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics. J Med Chem 65, 3420–3433 (2022). Zhang, S., O’Regan, R. & Xu, W. The emerging role of mediator complex subunit 12 in tumorigenesis and response to chemotherapeutics. Cancer 126, 939–948 (2020). 195 Zhang, Z., Klatt, A., Gilmour, D. S. & Henderson, A. J. Negative elongation factor NELF represses human immunodeficiency virus transcription by pausing the RNA polymerase II complex. J Biol Chem 282, 16981–16988 (2007). Zhang, Z., Lu, Y., Qi, Y., Xu, Y., Wang, S., Chen, F., Shen, M., Chen, M., Chen, N., Yang, L., Chen, S., Wang, F., Su, Y., Hu, M. & Wang, J. CDK19 regulates the proliferation of hematopoietic stem cells and acute myeloid leukemia cells by suppressing p53-mediated transcription of p21. Leukemia 36, 956–969 (2022). Zhu, J., Gaiha, G. D., John, S. P., Pertel, T., Chin, C. R., Gao, G., Qu, H., Walker, B. D., Elledge, S. J. & Brass, A. L. Reactivation of latent HIV-1 by inhibition of BRD4. Cell Rep 2, 807–816 (2012). Zhu, Q., Yu, T., Gan, S., Wang, Y., Pei, Y., Zhao, Q., Pei, S., Hao, S., Yuan, J., Xu, J., Hou, F., Wu, X., Peng, C., Wu, P., Qin, J. & Xiao, Y. TRIM24 facilitates antiviral immunity through mediating K63-linked TRAF3 ubiquitination. J Exp Med 217, e20192083 (2020). Zila, V., Müller, T. G., Müller, B. & Kräusslich, H.-G. HIV-1 capsid is the key orchestrator of early viral replication. PLoS Pathog 17, e1010109 (2021).
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Sadowski, Ivan
Characterization and exploitation of host factors to modulate HIV-1 latency
Text