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Elucidating the mechanism of reading frame selection by a viral internal ribosome entry site Au, Hilda Hiu Tung 2016

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 ELUCIDATING THE MECHANISM OF READING FRAME SELECTION BY A VIRAL INTERNAL RIBOSOME ENTRY SITE  by  Hilda Hiu Tung Au  B.Sc., The University of British Columbia, 2010  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)  December 2016  © Hilda Hiu Tung Au, 2016  ii  ABSTRACT  The Dicistroviridae intergenic region internal ribosome entry site (IGR IRES) exhibits the remarkable ability to bind the conserved core of the ribosome with high affinity. By mimicking the conformation of a tRNA, the IGR IRES can bypass the requirement for canonical initiation factors and Met-tRNAi,  and initiate translation from a non-AUG start codon in the ribosomal A site. The pseudoknot (PKI) domain of the IRES engages the decoding center upon initial ribosome binding, and subsequently translocates into the P site to allow delivery of the incoming aminoacyl-tRNA. Within the P site, the IRES adopts a conformation that is reminiscent of a P/E hybrid state tRNA to effectively co-opt the canonical elongation cycle. How the IGR IRES establishes the translational reading frame in the absence of initiation factors remains an outstanding question. Here, we elucidate the mechanism of reading frame selection by performing mutagenesis and biochemical assays to explore the function of specific IRES structural elements. We demonstrate that constituents of the Cricket paralysis virus PKI domain, including the helical stem, anticodon:codon-like base-pairing, and the variable loop region are optimized for IRES-mediated translation. Additionally, we reveal through extensive structural and biochemical studies that stem-loop III of the Israeli acute paralysis virus (IAPV) IRES mimics the acceptor stem of tRNA and functions in supporting efficient 0 frame translation. Finally, we established an infectious chimeric clone to investigate how translational regulation by the IAPV IRES affects the viral life cycle. Studies using this chimera demonstrate that formation of stem-loop VI upstream of the IAPV IRES contributes to optimal IRES activity and viral yield. Our findings suggest that extensive and complete tRNA-mimicry by the IAPV IGR IRES facilitates IRES-mediated translation and reading frame selection. iii  PREFACE  A portion of the Introduction has been published, and is adapted for use in this thesis: Au, H.H. and Jan, E. (2014). Novel viral translation strategies. Wiley Interdisciplinary Review RNA. 2014. I wrote this review with guidance from Dr. Eric Jan.     A version of Chapter 2 has been published: Au, H.H. and Jan, E. (2012). Insights into factorless translational initiation by the tRNA-like pseudoknot domain of a viral IRES. PLoS One. I conducted the experiments and performed the data analysis. The manuscript was written with guidance from Dr. Eric Jan.   A version of Chapter 3 has been published: Au, H.H., Cornilescu, G., Mouzakis, K.D., Ren, Q., Burke, J.E., Lee, S., Butcher, S.E., and Jan, Eric. (2015). Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection. Proceedings of the National Academy of Science. Data from Figures 3.2, 3.3, 3.6 and 3.7 have been contributed by Seonghoon Lee and Qian Ren. Data from Figures 3.12 and 3.13 have been contributed by our collaborators, Dr. Samuel Butcher, Gabriel Cornilescu, Kathryn Mouzakis and Jordan Burke. I wrote the manuscript with guidance from Dr. Eric Jan.   A version of Chapter 4 is currently under preparation for publication.    iv  TABLE OF CONTENTS Abstract ........................................................................................................................................................ ii Preface ......................................................................................................................................................... iii Table of Contents ....................................................................................................................................... iv List of Figures ............................................................................................................................................ vii List of Abbreviations ................................................................................................................................. ix Acknowledgements .................................................................................................................................. xiv Chapter 1: Introduction ............................................................................................................................. 1 1.1 Eukaryotic translation .................................................................................................................. 1 1.1.1 Translation initiation ................................................................................................... 1 1.1.2 Translation elongation ................................................................................................ 5 1.1.3 Transfer RNA and protein synthesis ........................................................................... 6 1.1.3.1 Structure of transfer RNA .............................................................................................. 6 1.1.3.2 Recognition of cogate transfer RNA during decoding ................................................... 6 1.2 Regulation of canonical translation initiation .............................................................................. 8 1.2.1 Phosphorylation status of eukaryotic initiation factor 2 ............................................. 9 1.2.2 Regulation of eukaryotic initiation factor 4E ............................................................ 10 1.3 Non-canonical translational mechanisms .................................................................................. 11 1.3.1 Translation initiation by internal ribosome entry ...................................................... 12 1.4 Recoding mechanisms ............................................................................................................... 17 1.4.1 Programmed -1 ribosomal frameshifts...................................................................... 18 1.4.2 Programmed +1 ribosomal frameshifts ..................................................................... 21 1.4.2.1 Yeast transposable elements ......................................................................................... 21 1.4.2.2 Antizyme is translated via +1 frameshift mechanism .................................................. 22 1.4.2.3 The role of suppressor transfer RNA and +1 frameshifting ......................................... 23 1.5 Dicistroviridae family of viruses ............................................................................................... 25 1.5.1 Genome organization ................................................................................................ 26 1.5.2 Viral-host interactions ............................................................................................... 29 1.5.3 Dicistroviridae intergenic region internal ribosome entry site ................................. 31 1.5.3.1 Mechanism of IGR IRES-mediated translation initiation ............................................ 33 1.5.3.2 IRES-mediated alternate reading frame selection ........................................................ 38 1.6 Thesis investigation ................................................................................................................... 40 Chapter 2: The integrity of the IGR IRES pseudoknot domain is important for IRES-mediated v  translation initiation ................................................................................................................................. 42 2.1 Introduction ............................................................................................................................... 42 2.2 Materials and methods ............................................................................................................... 43 2.2.1 DNA constructs ......................................................................................................... 43 2.2.2 In vitro transcription and translation ......................................................................... 44 2.2.3 Purification of 40S and 60S ribosomal subunits ....................................................... 44 2.2.4 Toeprinting/primer extension analysis ...................................................................... 45 2.3 Results ....................................................................................................................................... 46 2.3.1 Regions of CrPV PKI can be substituted by sequences from the prolyl-tRNA  anticodon stem-loop .................................................................................................. 46 2.3.2 A single base-pair deletion is tolerated in the PKI helical stem ............................... 51 2.3.3 An optimal length of the variable loop region (VLR) is required to maintain robust  IRES-mediated translation ........................................................................................ 53 2.3.4 Sequence identity of the VLR impacts IRES-mediated translation .......................... 59 2.3.5 IRES-mediated translation initiation is permissible at an adjacent and overlapping  alternate start site ...................................................................................................... 62 2.4 Discussion .................................................................................................................................. 66 Chapter 3: Global shape mimicry of tRNA of the Israeli acute paralysis virus intergenic region internal ribosome entry site mediates translational reading frame selection ...................................... 72 3.1 Introduction ............................................................................................................................... 72 3.2 Materials and methods ............................................................................................................... 75 3.2.1 Plasmids and constructs ............................................................................................ 75 3.2.2 In vitro translation assays.......................................................................................... 75 3.2.3 Purification of 40S and 60S ribosomal subunits ....................................................... 75 3.2.4 Toeprinting/primer extension assay .......................................................................... 76 3.2.5 Reconstitution of IRES-mediated translation ........................................................... 77 3.2.6 RNA structural probing............................................................................................. 77 3.3 Results ....................................................................................................................................... 78 3.3.1 The integrity of SLIII is important for IRES-mediated translation .......................... 78 3.3.2 Ribosome positioning is unaffected in SLIII mutants .............................................. 81 3.3.3 A6554 and A6576 contribute to IRES-mediated translation and reading frame  selection .................................................................................................................... 84 vi  3.3.4 Structural probing analysis of mutant ΔA6554 IRES ............................................... 85 3.3.5 Position of translocated ribosomes on the IAPV IRES ............................................ 88 3.3.6 Readout of eukaryotic release factor 1-dependent toeprints of IRES/ribosome  complexes ................................................................................................................. 94 3.3.7 Structural determination of the IAPV PKI domain................................................... 96 3.4 Discussion .................................................................................................................................. 98 Chapter 4: An adjacent stem-loop in honey bee dicistroviruses promotes IRES-mediated translation and virus infection ............................................................................................................... 105 4.1 Introduction ............................................................................................................................. 105 4.2 Materials and methods ............................................................................................................. 106 4.2.1 Reporter constructs ................................................................................................. 106 4.2.2 RNA structural probing........................................................................................... 106 4.2.3 Purification of 40S and 60S ribosomal subunits ..................................................... 107 4.2.4 Toeprinting/ primer extension analysis ................................................................... 107 4.2.5 Construction of the CrPV/IAPV chimeric infectious clone .................................... 108 4.2.6 In vitro transcription and translation ....................................................................... 108 4.2.7 Cell culture .............................................................................................................. 109 4.2.8 RNA transfection .................................................................................................... 109 4.2.9 Virus infection ........................................................................................................ 109 4.2.10 Western blotting ...................................................................................................... 109 4.2.11 Northern blotting ..................................................................................................... 110 4.2.12 Viral titres ............................................................................................................... 110 4.3 Results ..................................................................................................................................... 110 4.3.1 Formation of SLVI can be disrupted by 5' or 3' mutations ..................................... 110 4.3.2 An intact SLVI contributes to proper positioning of the ribosome ........................ 113 4.3.3 A CrPV/IAPV chimeric clone is infectious in Drosophila S2 cells ....................... 115 4.3.4 Disruption of SLVI reduces viral yield due to suboptimal IRES translation ......... 120 4.4 Discussion ................................................................................................................................ 121 Chapter 5: Conclusions and future directions ..................................................................................... 127 References ................................................................................................................................................ 135 Appendices ............................................................................................................................................... 148 Appendix A .......................................................................................................................................... 148 vii  Appendix B ........................................................................................................................................... 152 Appendix C ........................................................................................................................................... 158  viii  LIST OF FIGURES Figure 1.1  Pathway of eukarotic cap-dependent translation initiation ......................................................... 2 Figure 1.2   Eukaryotic initiation factor 2 kinases and recycling of eIF2·Met-tRNAi·GTP ternary complex by eIF2B .................................................................................................................................. 4 Figure 1.3   Structure of tRNA ...................................................................................................................... 7 Figure 1.4   Classification of viral internal ribosome entry sites ................................................................ 14 Figure 1.5   Programmed ribosomal frameshifts ......................................................................................... 19 Figure 1.6   Genome organization of Dicistroviridae ................................................................................. 27 Figure 1.7   Dicistroviridae intergenic region internal ribosome entry site ................................................ 34 Figure 1.8   Pathway of IGR IRES-mediated translation initiation ............................................................ 36 Figure 1.9   Identification of a novel overlapping reading frame in a subset of dicistroviruses ................. 39  Figure 2.1  Chimeric IRESs containing prolyl-tRNA and wild-type CrPV PKI regions ............................ 47 Figure 2.2   Translational activities of chimeric and mutant IRESs ........................................................... 49 Figure 2.3   The IRES helical stem tolerates bulges and a 1 base-pair deletion ......................................... 52 Figure 2.4   Toeprinting analysis of IRESs bearing mutations in the helical stem ..................................... 54 Figure 2.5   Alterations in the length of the variable loop region (VLR) adversely affect IRES-mediated translation ................................................................................................................................ 55 Figure 2.6   Toeprinting analysis of VLR insertion/ deletion mutant IGR IRESs ...................................... 57 Figure 2.7   The conserved A6205 is important for IRES-mediated translation ......................................... 60 Figure 2.8   Toeprinting/ primer extension assay of VLR point mutants.................................................... 61 Figure 2.9   IGR IRES-mediated translation initiation at an alternate translational start site ..................... 64 Figure 2.10 IRES translational activities of reporter constructs containing alternate translational start  sites .......................................................................................................................................... 65  Figure 3.1   Secondary structure of the IAPV IGR IRES ........................................................................... 74 Figure 3.2   Translational activities of IAPV IRES PKI mutants ............................................................... 79 Figure 3.3   The integrity of stem-loop (SL) III is important for 0 frame translation ................................. 80 Figure 3.4   Translational activities of helix P3.3 mutants .......................................................................... 82 Figure 3.5   Toeprinting/ primer extension analysis of helix P3.3 mutants ................................................ 83 Figure 3.6   Summary of SHAPE analysis of wild-type and ΔA6554 IRESs in solution ........................... 86 Figure 3.7   SHAPE probing analyses of wild-type and ΔA6554 IRESs in solution and bound to the ribosome ................................................................................................................................ 87 ix  Figure 3.8   DMS probing analyses of wild-type and ΔA6554 IRESs ........................................................ 90 Figure 3.9   Reconstitution of IRES-mediated translation restricted to the +1 frame ................................. 92 Figure 3.10  Pathway of IGR IRES-mediated translation initiation ........................................................... 93 Figure 3.11  Reconstitution of translocation using eukaryotic release factor 1 (eRF1) .............................. 95 Figure 3.12  Structure of the IAPV PKI domain ......................................................................................... 97 Figure 3.13  Docking of the IAPV PKI domain.......................................................................................... 99  Figure 4.1   Secondary structure of the IAPV IGR IRES and cognate stem-loop (SL) VI mutants ......... 111 Figure 4.2   Dimethyl sulfate (DMS) probing of SLVI mutants ............................................................... 114 Figure 4.3   Disruption of SLVI formation impairs ribosome positioning on the IAPV IRES ................. 116 Figure 4.4   The chimeric IAPV/CrPV virus is infectious in Drosophila S2 cells ................................... 118 Figure 4.5   Fusion of SLVI with the upstream cistron impairs structural protein synthesis .................... 119 Figure 4.6   Disruption of SLVI formation decreases viral yield .............................................................. 122     x  LIST OF ABBREVIATIONS aa-tRNA aminoacyl-tRNA ABPV  acute bee paralysis virus ASL  anticodon stem-loop ATP  adenosine triphosphate cDNA  complementary deoxyribonucleic acid CrPV  Cricket paralysis virus cryo-EM cryo-electron microscopy DMS  dimethyl sulfate DTT  dithiothreitol 4E-BP  eukaryotic initiation factor 4E-binding protein eEF  eukaryotic elongation factor eIF  eukaryotic initiation factor EMCV  encephalomyocarditis virus eRF  eukaryotic release factor FLuc  firefly luciferase GCN2  general control non-derepressible-2 GTP  guanosine triphosphate  HaIV  Halastavi arva virus HCV  hepatitis C virus h p.i.  hours post-infection h p.t.  hours post-transfection xi  HRI  heme-regulated inhibitor kinase IAPV  Israeli acute paralysis virus IGR  intergenic region IRES  internal ribosome entry site ITAF  IRES trans-acting factor KBV  Kashmir bee virus KOAc  potassium acetate m6A  N6-methyladenosine MgOAc magnesium acetate mRNA  messenger ribonucleic acid mTOR  mammalian target of rapamycin mTORC mammalian target of rapamycin complex NMIA  N-methylisatoic anhydride NMR  nuclear magnetic resonance ORFx  open reading frame x PABP  poly(A) binding protein PDCD4  programmed cell death 4 PERK  protein kinase RNA-like endoplasmic reticulum kinase PIC  preinitiation complex PKR  protein kinase RNA PK  pseudoknot PRF  programmed ribosomal frameshift xii  PSIV  Plautia stali intestine virus PV  poliovirus RdRp  RNA-dependent RNA polymerase RhPV  Rhopalosiphum padi virus RLuc  Renilla luciferase RRL  rabbit reticulocyte lysate rp  ribosomal protein rRNA  ribosomal ribonucleic acid S6K  S6 kinase SAFA  Semi-Automated Footprinting Analysis SAXS  small angle X-ray scattering Sf21  Spodoptera frugiperda SG  stress granule SHAPE  selective 2' hydroxyl acylation analyzed by primer extension siRNA  small interfering ribonucleic acid SL  stem-loop TLS  tRNA-like structure tmRNA  transfer-messenger ribonucleic acid tRNA  transfer ribonucleic acid TSV  Taura syndrome virus 5'-UTR  5'-untranslated region VLR  variable loop region xiii  VPg  viral protein genome-linked WT  wild-type   xiv  ACKNOWLEDGEMENTS  First and foremost, I would like to thank my supervisor, Dr. Eric Jan, for providing me with the opportunity to work in his laboratory. I am extremely grateful for his guidance and mentorship throughout my graduate career, and for providing me with many opportunities to explore my research interests and to establish myself as an independent scientist. His passion for research has been inspirational. I would also like to extend my thanks to the members of my supervisory committee, Dr. George Mackie, Dr. Thibault Mayor, and Dr. Marc Horwitz for their expertise, insights and constructive feedback.   I am grateful to have had the opportunity to work with many talented post-doctoral fellows and graduate students. Thank you for creating an intellectually stimulating environment, for all the thought-provoking discussions, and more importantly, the encouragement and support you have extended during times where I have struggled to find motivation - my experience in graduate school would not be the same without you.  I would also like to thank the National Sciences and Engineering Council of Canada (NSERC) and the University of British Columbia for providing financial support and funding.    Finally, I would like to express my deepest gratitude to my family for their unconditional love and support. Thank you for believing in me and being my pillar of support during difficult times. This is dedicated to you.       1  CHAPTER 1: INTRODUCTION 1.1 Eukaryotic translation 1.1.1 Translation initiation  Protein synthesis, which includes initiation, elongation, termination, and ribosome recycling, represents an essential process that mediates the transfer of genetic information encoded within a messenger RNA (mRNA) into its nascent polypeptide. In eukaryotes, initiation of translation of the majority of mRNAs occurs via a cap-dependent scanning mechanism that involves the concerted action of at least 12 eukaryotic initiation factors (eIFs) (reviewed in (Hinnebusch et al., 2016; Hinnebusch and Lorsch, 2012)). The 5' cap and 3' poly(A) tail are salient hallmarks of eukaryotic mRNAs that have intrinsic roles during this process (Figure 1.1). The cap moiety consists of a 7-methyl-guanosine that is linked to the first nucleoside of mRNA by an inverted 5'-5' triphosphate bridge (reviewed in (Shuman, 2002)). The cap is added co-transcriptionally to nascent mRNAs and functions to protect the RNA from degradation by exonucleases and promote pre-mRNA splicing (Edery and Sonenberg, 1985; Konarska et al., 1984; Lewis and Izaurralde, 1997; Schwer et al., 1998). Additionally, the 5' cap is recognized by the nuclear cap-binding complex which supports a pioneer round of translation important for mRNA quality control (reviewed in (Maquat et al., 2010)). During translation initiation, the 5' cap binds the cap-binding complex, eIF4F, which is comprised of three factors: the cap-binding protein, eIF4E; the RNA helicase, eIF4A; and the adaptor protein, eIF4G. eIF4G serves as an important scaffold for ribosome recruitment and mediates circularization of the transcript through interaction with poly(A) binding protein (PABP) bound to the mRNA 3' poly(A) tail. The initiation process begins by formation of the 43S preinitiation complex (PIC), which consists of the 40S ribosomal subunit, the ternary complex eIF2·Met-tRNAi·GTP, eIF1, eIF1A, eIF3 and eIF5 (reviewed in (Hinnebusch et al., 2016; Hinnebusch and Lorsch, 2012)). Through interaction of eIF3 with eIF4G (in the cap-binding complex), the 43S PIC is recruited proximally to the 5' cap and subsequently undergoes directional scanning for the authentic initiation  2    Figure 1.1 Pathway of eukaryotic cap-dependent translation initiation. The 5' 7-methyl-guanosine cap of cellular mRNA is bound by the cap-binding complex eIF4F, which consists of the cap-binding protein 4E, the helicase 4A, and the scaffold protein 4G. eIF4G facilitates recruitment of the 43S preinitiation complex (1) and circularization of the mRNA through interaction with poly(A) binding proteins (PABPs) bound to the 3' poly(A) tail. Following 43S recruitment, the complex undergoes ATP-dependent directional scanning (2) to locate the AUG start codon within a favorable context. (3) Start codon recognition and anticodon:codon pairing results in hydrolysis of the eIF2-bound GTP in a process mediated by eIF5. Subsequently, eIF5B mediates joining of the 60S ribosomal subunit to form an elongation-competent 80S ribosome (4). Reproduced with permission from (Au and Jan, 2014).   3  codon. Scanning is facilitated by the helicase, eIF4A, which unwinds local RNA secondary structure in an ATP-dependent manner, together with the factors, eIF1 and eIF1A, which induce a scanning-competent, 'open' conformation of the ribosome (Passmore et al., 2007). To ensure fidelity in translation initiation, the scanning complex can discriminate against and bypass initiation codons in poor sequence context and/or near-cognate triplets, and assemble at the correct initiation codon. Translation typically initiates at an initiation codon that is located within an optimal Kozak consensus sequence of GCC(A/G)CCAUGG, where a purine is at the -3 position and a guanine is at the +4 position (relative to the A of the AUG codon, designated as +1) (Kozak, 1986, 1991). Start codon recognition is characterized by complementary Watson-Crick base-pairing between the anticodon of Met-tRNAi and the AUG codon in the ribosomal P site.  Establishment of anticodon: codon base-pairing triggers the displacement of eIF1(Maag et al., 2005), and the ribosomal complex consequently adopts a scanning-incompetent, 'closed' conformation that is engaged on the mRNA (Passmore et al., 2007).  The arrested PIC commits to initiation at the start codon through the activity of eIF5, a GTPase-activating protein, which stimulates the hydrolysis of eIF2-bound GTP (Paulin et al., 2001). GTP hydrolysis reduces the affinity of eIF2 to Met-tRNAi, thus resulting in dissociation of eIF2-GDP from the 40S ribosomal subunit (Kapp and Lorsch, 2004). Release of the remaining initiation factors eIF1, eIF1A and eIF3 is facilitated by eIF5B, which promotes joining of the 60S ribosomal subunit (Pestova et al., 2000). Upon completion of the translation initiation cycle, an elongation-competent 80S ribosome is positioned at the translational start site, with the Met-tRNAi base-paired with the start codon in the P site. The adjacent A site is vacant to allow the ribosome to sample for, and accommodate the next aminoacyl-tRNA. Because the eIF2·Met-tRNAi·GTP ternary complex represents a key factor during initiation, the guanine nucleotide exchange factor, eIF2B, recycles the eIF2-bound GDP to GTP, thus replenishing the ternary complex pool for subsequent rounds of translation initiation (Figure 1.2) (Gomez et al., 2002; Gomez and Pavitt, 2000).   4     Figure 1.2 Eukaryotic initiation factor 2 kinases and recycling of eIF2·Met-tRNAi·GTP ternary complex by eIF2B.  During translation initiation, the eIF2·Met-tRNAi·GTP ternary complex is recruited to the 43S preinitiation complex and is involved in start codon recognition (1). Upon cognate codon:anticodon pairing, eIF5 mediates hydrolysis of eIF2-bound GTP to GDP (2), which must be recycled by the guanine nucleotide exchange factor eIF2B for subsequent rounds of initiation (3). The α-subunit of eIF2 is susceptible to phosphorylation by various eIF2α kinases (PERK, HRI, PKR, and GCN2) in response to specific environmental triggers. The phosphorylated form of eIF2α acts as a competitive inhibitor of eIF2B (4), thus preventing recycling of the GDP-bound eIF2 to the GTP-bound form and decreasing the availability of the ternary complex pool for translation initiation. Some factors such as eIF2A/2D have Met-tRNAi delivering activities and may be used in specialized translation initiation (5). Reproduced with permission from (Au and Jan, 2014).  5  1.1.2 Translation elongation   Following initiation, translation enters into an elongation phase, which requires the activity of two primary factors, eukaryotic elongation factors (eEF) 1A and eEF2 (Dever and Green, 2012). eEF1A binds and delivers aminoacyl-tRNAs (aa-tRNAs) in a ternary complex consisting of aa-tRNA·GTP·eEF1A for sampling of codon:anticodon pairs in the A site. Codon:anticodon recognition results in small ribosomal subunit domain closure and distortion of the tRNA to achieve the 'A/T' state so that interactions with both the decoding center and eEF1A can be simultaneously maintained (Valle et al., 2002). Ribosome binding induces conformational changes in eIF1A's domain 2 that result in its interaction with small subunit rRNA (Schmeing et al., 2009). Additionally, loss of stabilizing interactions between tRNA and the switch I domain of eIF1A triggers activation of the GTPase center (Schmeing et al., 2009), hydrolysis of eEF1A-bound GTP, and subsequent release of eIF1A (reviewed in (Dever and Green, 2012)). Dissociation of eEF1A provides an unobstructed path for the tRNA's acceptor end to be accommodated into the peptidyl transferase center of the large subunit. Peptidyl transfer occurs rapidly, leading to peptide bond formation with the adjacent P-site peptidyl-tRNA catalyzed by ribosomal RNA via a substrate-assisted mechanism (Schmeing et al., 2005). During catalysis, the target carbonyl-carbon of peptidyl-tRNA becomes reoriented and exposed to facilitate nucleophilic attack by the α-amino group of the neighbouring aminoacyl-tRNA (Schmeing et al., 2005). In the absence of an appropriate A-site ligand, the induced-fit mechanism sequesters the carbonyl-carbon in an inaccessible conformation to preclude nucleophilic attack by water, thus preventing hydrolysis of the polypeptide (Schmeing et al., 2005). Peptide bond formation leads to spontaneous ratcheting of the ribosome (Frank and Agrawal, 2000), which displaces the tRNAs into intermediate conformational states, also called 'P/E' and 'A/P' hybrid states (Moazed and Noller, 1989). The hybrid state is characterized by the relative movement of the tRNA acceptor stems into the adjacent site, despite the anticodon stem-loops remaining in the canonical sites. The pre-translocation state of the ribosome is recognized by the translocase, eEF2 (Dorner et al., 2006; Semenkov et al., 2000). Association of eEF2 and hydrolysis of its bound GTP catalyzes the translocation event, moving the P/E and A/P hybrid-state tRNAs into their classical states in the E and P 6  sites (Frank and Agrawal, 2000; Frank et al., 2007; Taylor et al., 2007a). In the post-translocation state, the deacylated-tRNA and peptidyl-tRNA occupy the E and P sites, respectively, leaving the A site vacant to receive an incoming aa-tRNA in the subsequent translocation cycle.   1.1.3 Transfer RNA and protein synthesis 1.1.3.1  Structure of transfer RNA  tRNAs play a central role in protein synthesis, as they serve as the adaptor molecule between the amino acid and the triplet codon that is 'read' during the decoding process. tRNAs adopt a conserved cloverleaf secondary structure that consists of several distinguishing features: the acceptor stem, which encompasses the 3' CCA trinucleotide; the anticodon arm, which base-pairs with the corresponding triplet during decoding; the D loop, which contains a tandem dihydrouridine modification; the TΨC loop, which contains a thymidine-pseudouridine-cytosine motif; and the variable (V) loop, which contains a varying number (3-21) of nucleotides (reviewed in (Redko et al., 2007)). A long-range kissing loop interaction between the D and TΨC loops enables the tRNA to adopt a characteristic L-shape conformation (Figure 1.3) (Quigley and Rich, 1976).  1.1.3.2   Recognition of cognate transfer RNA during decoding  Discrimination against non-cognate tRNAs is essential in the decoding process to ensure that the appropriate amino acid is added to the growing polypeptide chain. Recognition of cognate tRNA is facilitated by interaction of 16S rRNA bases A1492 and A1493 (or A1755 and A1756 for eukaryotic 18S rRNA), which flip out of the internal loop of helix 44 upon binding of cognate tRNA binding, with the minor groove of the codon-anticodon helix (Ogle et al., 2001). Within their new conformations, A1493 and A1492 can interact with the first and second base-pairs of the codon-anticodon helix, respectively. Additionally, the universally conserved G530 of 16S rRNA (or G577 in eukaryotes) interacts with the second position of the anticodon and the third position of the codon (Ogle et al., 2001). Altogether, these  7      Figure 1.3 Structure of tRNA. (A) The cloverleaf secondary structure of tRNAs consists of the acceptor stem, which encompasses the 3' CCA trinucleotide; the anticodon arm, which base-pairs with the corresponding triplet during decoding; the D loop, which contains a tandem dihydrouridine modification; the TΨC loop, which contains a thymidine-pseudouridine-cytosine motif; and the variable (V) loop. (B) The tertiary L-shape conformation of tRNA is mediated by kissing loop interaction between the D and TΨC loops. Reproduced with permission from (Hori, 2014).   8  interactions facilitate the ribosome in discriminating between true Watson-Crick base-pairs and mismatches in the first two base-pairs of the codon-anticodon helix. The minor groove of the third base-pair is not as stringently monitored by the ribosome and can accommodate other base-pairing geometries, thus allowing non-canonical base-pairs at the 'wobble' position (Ogle et al., 2001). Cognate tRNA binding also induces conformational changes in the small ribosomal subunit involving rotation of the 40S head domain toward the large subunit, which promote closure of the small subunit around the A site (Ogle et al., 2002). While these conformational changes are not observed for near- and non-cognate tRNA binding, they can be recapitulated in the presence of paromomycin, an aminoglycoside antibiotic (Carter et al., 2000; Ogle et al., 2002). Paromomycin binds to the major groove of helix 44 and partially induces the structural rearrangements that are characteristic of the closed small subunit conformation (Ogle et al., 2002). The presence of paromomycin also constrains A1492 and A1493 such that they are in the flipped orientation, independently of cognate codon-anticodon base-pairing (Carter et al., 2000; Ogle et al., 2002). Thus, by stabilizing the extruded positions of A1492 and A1493, paromomycin (and other aminoglycosides) reduces the energetic penalty of non-cognate tRNA binding and increases the affinity of tRNA for the A site (Carter et al., 2000). This is consist with the established properties of paromomycin in promoting miscoding during translation.    1.2 Regulation of canonical translation initiation  Translation is highly regulated, especially in cells that undergo environmental stress, in organismal development, in enucleate cells like red blood cells and during virus infection. Because a substantial amount of the cell's resources and energy are expended towards protein synthesis, translation has to be controlled in order to maintain cellular homeostasis. During acute stress, such as virus infection or heat shock, overall protein synthesis is repressed. Translational control enables coordinated changes in gene expression to restore homeostasis, or in some instances, to induce apoptosis. Furthermore, translational control enables the cell to precisely modulate the critical concentrations of proteins. It is now apparent that translation initiation is subject to most of translational regulation, as it provides a rapid and 9  reversible means to control protein expression; however, translation can also be regulated at the elongation step. One way by which this is achieved is through modification of eEF2. Domain IV of eEF2, which contacts the 40S decoding center, is critical for its ability to mediate translocation (Davydova and Ovchinnikov, 1990; Nygard and Nilsson, 1990; Spahn et al., 2004b; Taylor et al., 2007b). The tip of domain IV bears a conserved histidine residue (His699 in S. cerevisiae eEF2) that can be post-translationally modified to diphthamide, a conserved modification that is non-essential for cell viability (reviewed in (Dever and Green, 2012)). Interestingly, diphthamide modification is targeted for ADP ribosylation by diphtheria toxin, resulting in eEF2 inactivation and subsequent inhibition of translation (reviewed in (Collier, 2001)). Regulation of translation is commonly achieved through pathways that sense and respond to cellular stress, and the consequent down-regulation of factors that orchestrate the canonical initiation pathway. Much effort has been invested into elucidating the molecular mechanisms that enable translational control. Two important targets of translational control, which will be discussed in further detail, include restricting the availability of the ternary complex by eIF2α phosphorylation and the regulation of eIF4E activity.  1.2.1 Phosphorylation status of eukaryotic initiation factor 2  The availability of the eIF2·Met-tRNAi·GTP ternary complex is critical in regulating translation initiation, as it mediates delivery of the Met-tRNAi to the initiation codon. Following start codon recognition and GTP hydrolysis, eIF2-GTP is regenerated by the activity of eIF2B which acts as a guanine nucleotide exchange factor (Figure 1.2). eIF2 is a trimeric factor that consists of α-, β- and γ-subunits. The γ-subunit mediates GTP and Met-tRNAi binding. The α-subunit is subject to reversible phosphorylation at Ser51. In its phosphorylated state, eIF2 binds with high affinity to eIF2B, which consequently impedes the ability of eIF2B to mediate recycling of GDP to GTP (Krishnamoorthy et al., 2001). Because the cellular concentration of eIF2 is in excess over eIF2B, even minute increases in phosphorylated eIF2 can significantly reduce the availability of the ternary complex, thus inhibiting global translation initiation (Rowlands et al., 1988). There are four mammalian protein kinases that 10  respond to specific cellular stresses and trigger eIF2α phosphorylation (reviewed in (Holcik and Sonenberg, 2005)): heme-regulated inhibitor kinase (HRI), which is activated upon heme deficiency; protein kinase RNA (PKR), which is activated by double-stranded RNAs; PKR-like endoplasmic reticulum kinase (PERK), which is activated by endoplasmic reticulum stress; and general control non-derepressible-2 (GCN2), which is activated upon amino acid depletion (Figure 1.2). Induction of eIF2α phosphorylation represents a major translational control mechanism that effectively attenuates global translation during diverse cellular stresses.  1.2.2 Regulation of eukaryotic initiation factor 4E  eIF4E plays a critical role in the canonical initiation pathway by bridging the interaction between the transcript's 5' cap and the 40S ribosomal subunit. eIF4E is subject to regulation by MAP kinase-interacting kinases (Mnks)-mediated phosphorylation upon activation of the p38 MAPK and MEK-ERK pathways (Fukunaga and Hunter, 1997; Waskiewicz et al., 1997). Mnks phosphorylate eIF4E at Ser209 near its C-terminus by binding to eIF4G (Flynn and Proud, 1995; McKendrick et al., 2001). The functional consequence of eIF4E phosphorylation remains unclear.   An alternate mechanism in regulation of eIF4E is to restrict its accessibility. The activity of eIF4E is primarily modulated by eIF4E-binding proteins (4E-BPs, designated EIF4EBP1-3 in mammals), which in turn are intricately regulated by phosphorylation events (reviewed in (Ma and Blenis, 2009; Richter and Sonenberg, 2005)). In the hypo-phosphorylated state, 4E-BP1 competes with eIF4G for a common binding site on eIF4E, which effectively abrogates the interaction of eIF4E with the scaffold protein. Activation of the kinase, mammalian target of rapamycin (mTOR), results in 4E-BP1 hyper-phosphorylation and the release of eIF4E to be assimilated into the cap binding complex for use in translation initiation. mTOR is a serine/threonine kinase that responds to environmental signals, including nutrient availability, cellular energy status, and growth factors or hormones, to regulate cell growth and proliferation. There are two functionally and structurally divergent species: mammalian TOR complex 11  (mTORC) 1 and 2 (Laplante and Sabatini, 2012). Despite sharing a common catalytic component, mTORC1 and mTORC2 are multi-factor complexes that have different components and substrate specificities. mTORC2, comprised of mTOR, Rictor, Protor and mSIN1, activates a subset of AGC kinases including serum/ glucocorticoid regulated kinase 1 and AKT, which regulate cell survival (Oh and Jacinto, 2011). mTORC1, which consists of mTOR, Raptor, PRAS40, Deptor, and mLST8, directly influences global protein synthesis by modulating components of the translational machinery. In addition to targeting 4E-BPs, mTORC1 also induces phosphorylation and activation of S6 kinases (S6K), S6K1 and S6K2, which consequently phosphorylate various substrates including ribosomal protein S6 (Banerjee et al., 1990), eIF4B (Raught et al., 2004) (which enhances the processivity of eIF4A (Rogers et al., 2002)), and programmed cell death 4 (PDCD4) (Dorrello et al., 2006). PDCD4 represses translation by binding to and inhibiting the helicase activity of eIF4A (Yang et al., 2003). S6K-mediated phosphorylation of PDCD4 targets it for proteasome-dependent degradation via the ubiquitin ligase β-TrCP (Dorrello et al., 2006); thus, eIF4A is available to engage in translation initiation (Dorrello et al., 2006; Yang et al., 2003). Because translation consumes substantial energy and cellular resources, various elaborate mechanisms have evolved to integrate environmental cues and to modulate global protein synthesis accordingly. mTOR-mediated regulation serves at the nexus of several major regulatory pathways, thereby signaling cells to grow and proliferate under favorable conditions, or conversely, to attenuate translation and promote cell survival when resources are limiting.  1.3 Non-canonical translational mechanisms  During conditions of stress, global translation is repressed as a means to conserve cellular energy and resources, and to facilitate reprogramming of gene expression. Paradoxically, translational control occurs selectively, whereby a subset of mRNAs evades translational repression and remains preferentially translated. Such transcripts utilize alternative, non-canonical translation mechanisms that bypass the conventional scanning mechanism or dispense with the need for factors utilized during cap-dependent 12  translation. One such example that will be discussed in further detail is translation initiation by internal ribosome entry.  1.3.1 Translation initiation by internal ribosome entry  Although cap-dependent translation initiation represents the primary pathway by which the majority of cellular transcripts recruits the translational machinery, an alternative, non-canonical mode of translation initiation occurs through internal ribosome entry. This translation mechanism involves cis-acting, generally structured RNA elements called internal ribosome entry sites (IRESs), which typically reside in the untranslated regions of viral genomes or in a subset of cellular mRNAs. IRES elements act in a 5' end-independent manner to recruit the translational machinery to an internal position of the transcript.   Historically, IRESs were first described in the picornaviruses, poliovirus (PV) and encephalomyocarditis virus (EMCV) (Jang et al., 1988; Pelletier and Sonenberg, 1988). The viral RNAs were initially scrutinized for their ability to mediate cap-independent translation due to the peculiarity that the picornavirus genome lacks a conventional 5' 7-methyl-guanosine cap. Instead, the 5'-end contains the genome-linked viral protein (VPg), and a highly structured 5'-untranslated region (5'-UTR) that harbours multiple non-initiating AUG codons. Because these properties deem the RNA unconducive to linear scanning by the preinitiation complex, translation initiation on these viral RNAs cannot be adequately explained by the canonical cap-dependent pathway. To experimentally demonstrate internal initiation, the viral 5'-UTR elements were inserted into the intercistronic space within the context of a bicistronic reporter construct and indeed, confirmed that internal ribosome binding and initiation were possible (Jang et al., 1988; Pelletier and Sonenberg, 1988). IRES elements were subsequently identified in other picornaviruses, including foot-and-mouth disease virus (Belsham and Brangwyn, 1990; Kuhn et al., 1990), human rhinoviruses (Borman and Jackson, 1992), and hepatitis A virus (Brown et al., 1994; Glass et al., 1993). Internal initiation by these viral IRESs was unambiguously demonstrated through the observation that an artificially-synthesized circularized RNA harboring the EMCV IRES can initiate translation, thus 13  proving that attachment of the translational machinery on these RNA elements occurs internally (Chen and Sarnow, 1995). Viral IRESs can be classified into four primary groups based on the requirement for canonical initiation factors, initiator Met-tRNAi and IRES trans-acting factors (ITAFs) (Kieft, 2008) (Figure 1.4). Group 4 IRESs, best exemplified by the poliovirus IRES, require the full complement of initiation factors except eIF4E, initiator-tRNA, and ITAFs for functionality. Initiation complexes assembled on Group 4 IRESs must undergo scanning to locate the initiation codon. Group 3 IRESs, exemplified by the EMCV and foot-and-mouth disease virus IRESs, exhibit the same factor dependence as Group 4 IRESs; however, these IRESs can directly position the ribosome at the translation start site. Group 2 IRESs, including the hepatitis C virus (HCV) and classical swine fever virus IRESs, can directly bind the 40S ribosomal subunit using the initiator Met-tRNAi and a substantially reduced subset of initiation factors that consists only of eIF3 and eIF2 (Pestova et al., 1998). Binding of the 40S subunit on an HCV-like IRES directly positions the initiation codon in the ribosomal P site without initial scanning by the preinitiation complex (Pestova et al., 1998). Although HCV-like IRESs utilize a conventional eIF2-dependent mode of initiation, an alternate eIF2-independent pathway has been observed (Kim et al., 2011; Pestova et al., 2008; Terenin et al., 2008). It has been reported that alternative factors, including eIF2A, eIF5B, Ligatin, and MCT-1/DENR can mediate efficient recruitment of Met-tRNAi to the binary IRES/40S complexes (Figure 1.2)(Kim et al., 2011; Pestova et al., 2008; Skabkin et al., 2010; Terenin et al., 2008). Furthermore, eIF2A undergoes redistribution from the nucleus to the cytoplasm during HCV infection, and its knockdown reduced viral infectivity (Kim et al., 2011). Interestingly, HCV infection induces phosphorylation of eIF2 and PKR to drastically inhibit cap-dependent translation (Garaigorta and Chisari, 2009; Kang et al., 2009). Although the physiological relevance remains to be confirmed, usage of such alternative factors that exhibit Met-tRNAi-delivering activity may allow HCV IRES-mediated translation to be refractory to the inhibitory effects of eIF2 phosphorylation, thus enabling efficient viral propagation. Group 1 IRESs, found within the intergenic region of members of the Dicistroviridae family, utilize the most streamlined mechanism of initiation. They dispense the need for all canonical initiation 14     Figure 1.4 Classification of viral internal ribosome entry sites. Viral internal ribosome entry sites can be classified into one of four groups. Group 4 IRESs require Met-tRNAi, trans-acting factors, and all canonical initiation factors with the exception of eIF4E. Ribosomes recruited by Group 4 IRESs must undergo scanning to initiate translation at a start codon downstream of the IRES. Group 3 IRESs exhibit the same factor dependence as those of Group 4, but can directly position the ribosome at the initiation site. Group 2 IRESs can directly bind the 40S ribosomal subunit and require only eIF2, eIF3 and Met-tRNAi. The IRESs of Group 1 utilize the most streamlined translation initiation mechanism that dispenses the need for all canonical initiation factors and Met-tRNAi. Reproduced with permission from (Plank and Kieft, 2012).   Group 4 Group 3 Group 2 Group 1 15  factors to directly bind and recruit ribosomes (Jan and Sarnow, 2002; Sasaki and Nakashima, 1999, 2000; Wilson et al., 2000a). Remarkably, unlike the cap-dependent pathway, Group 1 IRES-mediated translation initiates from the ribosomal A site and at a non-AUG start codon (Jan and Sarnow, 2002; Sasaki and Nakashima, 1999, 2000; Wilson et al., 2000a) .  In general, IRESs utilize only a subset of the canonical translation initiation factors and may require auxiliary proteins or ITAFs that are not normally involved in cap-dependent translation, but are usurped for IRES-mediated translation. IRES elements vary in their requirement for ITAFs, which are thought to promote remodeling of RNA structure to stabilize a specific, active conformation of the IRES (reviewed in (Plank and Kieft, 2012)). For example, the poliovirus IRES exhibits minimal activity in a rabbit reticulocyte lysate translation extract (Pelletier et al., 1988), and is only efficiently translated upon supplementation with HeLa cytoplasmic extracts (Dorner et al., 1984). This observation provided the initial evidence that specific trans-acting factors are required for optimal activity of some IRESs. Because of the reduced requirement for initiation factors, IRES-dependent translation can be active or stimulated under conditions such as virus infection and cellular stress, when the activities of specific targeted translation factors are compromised. For example, PV infection results in global translational repression through targeted degradation of host initiation factors, eIF4G and PABP, by the 2Apro and 3Cpro viral proteases (Etchison et al., 1982; Gradi et al., 1998; Joachims et al., 1999; Krausslich et al., 1987). Cleavage of eIF4G by 2Apro severs the eIF4E and eIF3 binding sites (Lamphear et al., 1995; Mader et al., 1995), thus abolishing de novo translation initiation; however, because eIF4E  is dispensable for PV IRES-mediated translation, the IRES can specifically recruit the carboxy-terminal cleavage product (encompassing the eIF3 and eIF4A binding sites) and retain translational activity (Ohlmann et al., 1996). Therefore, viral IRESs can effectively hijack the host translational machinery to mediate preferential and/or an exclusive switch from a cap-dependent to viral IRES-mediated mode of translation. Host translational shutoff is an advantageous strategy for viral protein synthesis, owing to the increase in the availability of ribosomes and translation factors that can be diverted towards viral RNA translation. 16    IRES elements have since been identified in a myriad of viral genomes and putatively in various cellular transcripts. The cellular IRES field has remained contentious and been undermined by much skepticism due to the lack of appropriate controls in validating putative candidates (Kozak, 2005; Shatsky et al., 2010). The controversy stemmed from caveats associated with the bicistronic reporter test, which - unless additional controls are performed - cannot discount the possibility of cryptic promoters or splice sites in generating monocistronic transcripts that can be readily expressed by cap-dependent translation (reviewed in (Kozak, 2003, 2005)); thus, candidates that seemingly exhibit intrinsic IRES activity have been falsely identified. Generally, validation of a putative IRES begins by assessing its translational activity when a free 5'-end is not accessible for recruitment of initiation factors. A common practice is to use the hallmark bicistronic reporter in which the putative IRES sequence is inserted into the intercistronic space between two reporters. Reporter expression can be examined using transcription-translation coupled extracts in vitro, or by transfection of the reporter construct into a suitable cell line. IRES activity can be gauged by an observed increase in the expression ratio of the downstream reporter to the upstream reporter (which serves as a normalizing control), relative to a bicistronic construct lacking the putative sequence. Alternatively, a monocistronic reporter construct can be used, provided that it harbors a stable 5'-proximal hairpin to block scanning preinitiation complexes (Macejak and Sarnow, 1991). Observed IRES activity may represent an artefact of monocistronic reporter transcripts that arise from cryptic promoters and/or splice sites and/or endonucleolytic cleavage (reviewed in (Thompson, 2012)), which is especially concerning when bicistronic DNA constructs are used. To control for this, Northern blotting can be performed to probe the presence of aberrant RNAs; however, if the monocistronic transcripts are generated at low frequency, the sensitivity of Northern blots may not allow these species to be readily detected. Additionally, siRNAs targeting the upstream reporter can be co-transfected with the bicistronic constructs to determine if a similar reduction in the expression of both reporters is observed, which is anticipated if the bicistronic reporter remains intact (Van Eden et al., 2004). Finally, usage of RNA bicistronic constructs has also been reported to circumvent issues associated with DNA reporter systems (reviewed in (Thompson, 2012)).  17   Recently, alternative modes of non-canonical translation, including N6-methyladenosine- and eIF3d-mediated initiation, have been identified (Lee et al., 2015; Lee et al., 2016; Meyer et al., 2015). Transcripts that bear N6-methyladenosine (m6A) modifications within their 5'-UTR can undergo cap-independent translation. The m6A residues act as ribosome engagement sites that can facilitate recruitment of the 43S preinitiation complex by direct interaction with eIF3 (Meyer et al., 2015). Because modified residues within the coding region cannot mediate initiation, m6A-induced translation occurs through a 5' end-dependent, rather than an internal entry-based mechanism (Meyer et al., 2015). eIF3 can also mediate cap-dependent translation of a subset of cell proliferation mRNAs by binding to an internal stem-loop in their 5' UTRs (Lee et al., 2015). eIF3d, a subunit of the eIF3 complex, adopts a cup-like architecture that confers cap-binding activity on specific capped RNAs (Lee et al., 2016). Interestingly, mRNAs translated via this mechanism encode cis-acting RNA elements that impede recruitment of the canonical cap-binding complex; thus, the use of multiple cap-dependent pathways can allow transcripts to be expressed under conditions where the canonical cap-binding protein eIF4E is inactivated. While these modes of translation may exhibit reduced factor requirement and/or mediate cap-independent translation, it is important to distinguish whether or not they represent bona fide IRES mechanisms based on their 5'-end dependence.  1.4 Recoding mechanisms    During elongation, accuracy in decoding is imperative to ensure that the encoded information is faithfully transmitted into the nascent polypeptide; however, under specific circumstances, it may prove to be advantageous if translational fidelity may be specifically modulated. Indeed, viruses have evolved various recoding mechanisms to increase the coding capacity within the constraints of their compact genomes. Such viral strategies include the use of alternative initiation sites, directing elongating ribosomes into alternate reading frames, and read-through of termination codons. Furthermore, recoding events observed may represent an additional mechanism to regulate translation and gene expression.   18  1.4.1 Programmed -1 ribosomal frameshifts  During the elongation phase of translation, the ribosome may be subjected to the effects of cis-acting RNA elements that shift the translational apparatus into alternate reading frames. So-called programmed ribosomal frameshift (PRF) signals may displace the elongating ribosome toward the 5'- (-1 PRF) or 3'-end (+1 PRF) (Figure 1.5). The parameters constituting a -1 PRF have been extensively defined in viral genomes and involve a 'slippery' heptanucleotide RNA motif with the consensus X_XXY_YYZ (with X, Y and Z designating three different nucleotides) and a downstream stimulating element (usually a pseudoknot, and in some cases, a stable hairpin) located six to eight nucleotides from the 3' boundary of the slippery sequence (Brierley et al., 1992; Dinman et al., 1991; Kim et al., 1999; Yu et al., 2011). A slippery sequence alone is insufficient for frameshifting to occur (Kontos et al., 2001; Tu et al., 1992). An additional stimulatory element is required to induce sufficient pausing of the elongating ribosome such that the interactions between the mRNA and the A- and P-site tRNAs are disrupted to allow translocation into an alternate frame (Brierley et al., 1989). Because only the wobble positions of the -1 frame codons are changed relative to the reference frame, -1 decoding involves re-pairing of the mRNA with near-cognate tRNAs. By reconstituting an active -1 frameshifting event using mammalian ribosomes and a variant of the coronavirus IBV frameshift signal, cryo-EM reconstructions of the stalled complexes provided significant insight into the conformational rearrangements and mechanical tensions that occur during the decoding of a ribosomal frameshift (Namy et al., 2006). Most notably, the stimulatory pseudoknot obstructs the entrance of the mRNA channel and induces a ratchet-like rearrangement that traps the eEF2 translocase in an orientation that precludes A-site tRNA binding (Namy et al., 2006). Additionally, the P-site tRNA undergoes structural deformation that results from a bending of the D-arm, suggesting that the distortable nature of the tRNA might be essential in the frameshifting process (Namy et al., 2006). These cryo-EM structures provide a mechanical explanation for frameshifting, wherein the ribosome attempting to undergo eEF2-catalyzed translocation is counteracted by the blockage of the mRNA channel and occlusion of the A site (Namy et al., 2006). This resistance  19     Figure 1.5 Programmed ribosomal frameshifts. Programmed ribosomal frameshifts (PRFs) act on an elongating ribosome during which amino acids are sequentially added to the growing polypeptide chain. Three types of PRFs, including the -1, +1, and -2 PRFs have been identified in various viral genomes. In the -1 PRF, the frameshift site is comprised of a heptanucleotide sequence with the consensus X_XXY_YYZ (where X represents any nucleotide, Y represents an A or U, Z represents A, C, or U, and the underscore designates the codons in the 0 frame) and a downstream spacer sequence. While the consensus for -1 PRFs is well characterized, the frameshift consensus in +1 PRF is more variable. -1 PRFs require a 3' stimulatory element whereas +1 PRFs depend on cis-elements that facilitate the displacement of the ribosome into an alternate frame. The -2 PRF recently identified in the arterivirus porcine reproductive and respiratory syndrome virus produces a trans-frame fusion (TF). The -2 PRF occurs at a conserved G_GUU_UUU sequence and is stimulated by a conserved CCCANCUCC motif located 11 nucleotides downstream of the shift site. The mechanism of this mode of frameshift has not been fully elucidated. Reproduced with permission from (Au and Jan, 2014).   20  distorts the P-site tRNA and places sufficient strain on the anticodon-codon interaction to cause their dissociation (Namy et al., 2006). Alleviation of this strain likely promotes realignment of the anticodon with the codon in the -1 direction and the occurrence of a frameshifting event (Namy et al., 2006).       Frameshift elements are essential in regulating the translation of proteins during the viral life cycle. In HIV-1 and other related retroviruses, the expression of the viral proteins, including the RNA-dependent DNA polymerase, is under the regulation of frameshift signals (Jacks et al., 1988a; Jacks et al., 1988b; Jacks and Varmus, 1985). Classical translation of the viral mRNA terminates at a stop codon to generate exclusively the GAG protein, which represents the precursor for the viral structural proteins. Via a -1 PRF approximately 200 nucleotides upstream of the GAG stop codon, the GAG-POL fusion is generated from which the viral enzymes are derived (reviewed in (Brierley and Dos Ramos, 2006)). The frameshifting frequency thus dictates the precise ratio of the viral structural and non-structural proteins, which is crucial to the assembly of an infectious virion. Deviation of this ratio results in a decrease in viral yield (Shehu-Xhilaga et al., 2001). Though initially described in retroviral genomes, PRFs have since been documented in many other viruses and cellular genes and are more prevalent than originally thought.   Bioinformatic algorithms have proven to be extremely powerful in identifying frameshift elements, some of which act through nonconventional mechanisms (Chung et al., 2008; Fang et al., 2012; Firth et al., 2009; Jagger et al., 2012; Loughran et al., 2011). For example, until recently, the utilization of -2 PRF is poorly documented in eukaryotes. Computational analysis of various genotype isolates of porcine reproductive and respiratory syndrome virus, a member of Arteriviridae family, revealed a region of increased conservation in the +1 reading frame encoding the viral nsp 2 protein, designated as nsp2TF (Fang et al., 2012). Mass spectrometric and biochemical analyses demonstrated definitively that translation of nsp2TF occurs via a -2 PRF and necessitates both a G_GUU_UUU motif at the shift site and a downstream CCCANCUCC for efficient ribosomal frameshifting (Fang et al., 2012). Interestingly, nsp2TF is partitioned to specific foci under infection and is excluded from replication structures where 21  nsp2 resides (Fang et al., 2012). The observation that nsp2TF frameshift mutants severely impair virus replication further substantiates the physiological role of nsp2TF during infection. While the precise role of nsp2TF is currently unknown, the novel mode of frameshifting adds to the complexity in discerning the coding potential of a compact viral genome. It is also known that sequences downstream of the -2 PRF signal can also mediate a -1 PRF to generate the two viral replicase precursor polyproteins (Brierley et al., 1987; den Boon et al., 1991). Thus, many regulatory mechanisms must be in place to ensure that appropriate partitioning of translating ribosomes occurs to allow specific viral proteins to be expressed at precise times during the viral life cycle.  1.4.2 Programmed +1 ribosomal frameshift 1.4.2.1 Yeast transposable elements  +1 programmed frameshift elements have been documented in the Ty1 and Ty3 retrotransposons of Saccharomyces cerevisiae. The Ty retrotransposon encodes the overlapping GAG and POL genes, and replicates via a retroviral-like mechanism (Boeke et al., 1985). The GAG gene encodes the protein constituents of  the virus-like particle, while POL has sequence homologies to the reverse transcriptase, integrase and proteases of retroviruses and functions to catalyze the conversion of Ty mRNA into double-stranded DNA for integration into the genome (Adams et al., 1987; Garfinkel et al., 1985; Hansen et al., 1992; Mellor et al., 1985). The Ty POL gene is expressed as a GAG-POL fusion via a +1 programmed ribosomal frameshift. For the Ty1 retrotransposon, frameshifting occurs at a CUU_AGG_C minimal sequence which consists of overlapping leucine codons (CUU and UUA) in the 0 and +1 reading frames (Belcourt and Farabaugh, 1990). Interestingly, there are no distinct isoaccepting species for these two codons in yeast, but instead, tRNAUAGLeu  has the capacity to decode all six leucine codons. A frameshift is caused by tRNA slippage into the +1 frame at the two leucine codons, which is enhanced by ribosome stalling at the AGG codon due to low-abundance of the corresponding tRNACCUArg (Belcourt and 22  Farabaugh, 1990). Thus, the mechanism of Ty1 frameshift is dependent on re-pairing of the tRNA with the adjacent codon in the +1 frame.   For the Ty3 retrotransposon, POL3 overlaps the terminal 38 nucleotides of GAG3 in the +1 reading frame. The programmed ribosomal frameshift occurs at the GCG_AGU_U heptanucleotide (depicted as the GAG3 coding sequence), which encodes a rare 0 frame serine codon (AGU). Unlike the Ty1 retrotransposon, Ty3 frameshifting does not involve tRNA slippage, but instead, the P site peptidyl-tRNA occludes the 3' adjacent base and causes decoding of the +1 frame codon (Farabaugh et al., 1993). Frameshifting is influenced by two elements: the occurrence of the rare codon induces ribosome pausing due to the low availability of the cognate tRNA, and the 5' region of a 14-nucleotide sequence distal to the frameshift site (the Ty3 stimulator) stimulates the frameshift event (Farabaugh et al., 1993; Guarraia et al., 2007).   1.4.2.2 Antizyme is translated via a +1 frameshift mechanism  Programmed ribosomal frameshifts are also utilized in the translational regulation of cellular mRNAs, most notably antizymes. Antizyme acts as a negative regulator of ornithine decarboxylase, which catalyzes the initial step in polyamine synthesis. Antizyme functions by binding to and targeting ornithine decarboxylase for proteasome-mediated degradation and by adversely affecting the uptake of polyamines (Mitchell et al., 1994; Murakami et al., 1992; Suzuki et al., 1994; Zhang et al., 2003). The majority of antizymes are encoded by two partially overlapping cistrons: the first cistron encompasses a termination codon that is 3' adjacent to the frameshift signal, while the second cistron, which encodes the functional core of the antizyme in the +1 frame, is translated as a trans frame fusion. The presence of a termination codon downstream of the shift site is crucial for antizyme expression (Matsufuji et al., 1995); however, despite conservation of the UGA triplet (reviewed in (Ivanov and Atkins, 2007)), mutation to other termination codons only moderately reduces frameshift frequency (Matsufuji et al., 1995). Mutational analysis suggests that frameshifting does not occur due to tRNA slippage but through 23  occlusion of the 5' nucleotide of the termination codon (Matsufuji et al., 1995). The efficiency of frameshifting is enhanced by surrounding sequences and the presence of a stimulatory pseudoknot downstream of the shift site (Matsufuji et al., 1995). A recent study of the S. cerevisiae orthologue of ornithine decarboxylase antizyme (OAZ1) demonstrated that polyamine sensing is mediated by the nascent antizyme polypeptide (Kurian et al., 2011). The regulatory element resides at the C-terminus of the OAZ1 polypeptide, and functions in cis to inhibit translation of its own mRNA by causing a pile-up of ribosomes when intracellular polyamines are scarce (Kurian et al., 2011). Under conditions when the metabolite is abundant, polyamine binding to the nascent antizyme sequesters its regulatory effect on the ribosome, thus allowing synthesis of the full-length polypeptide (Kurian et al., 2011). Antizyme expression is therefore autoregulated by a sensor module that is encoded in the nascent polypeptide.      1.4.2.3 The role of suppressor transfer RNA and +1 frameshifting  tRNAs do not act as passive adaptors during the decoding process. Some tRNA species, termed frameshift suppressor tRNAs, can functionally suppress mutations that alter the translational reading frame. One early model, which proposed that that expansion of the anticodon by 1 nucleotide can promote quadruplet translocation for reading frame restoration (Yourno and Tanemura, 1970) gained traction with the discovery that the sufD suppressor contains a nucleotide insertion in its anticodon (Riddle and Carbon, 1973). It was subsequently generalized as the 'yardstick model', which postulates that the anticodon dictates the length of the translocation event. However, subsequent characterization of suppressor tRNAs, including the bacterial sufA6 and sufB2 suppressors, suggest that most do not adhere to this model. For sufA6 and sufB2, which each bear an additional nucleotide in the anticodon, tRNA modification likely occludes a 4 base-pair interaction with the codon and frameshifting instead occurs due to tRNA slippage (Qian et al., 1998).   Crystal structures of the anticodon stem-loops (ASLs) of +1 frame suppressor tRNAs bound to the ribosome have revealed how expanded anticodon loops can be accommodated into the decoding 24  center. Unlike canonical ASLs, the ASLCCCG and ASLACCC, which contain a base insertion at position 33.5 between positions 33 and 34, only engage in Watson-Crick interactions at the first two base-pairs of the anticodon:codon helix (Dunham et al., 2007). The third base of the anticodon forms nonstandard interactions with the wobble and/or the fourth base of the codon, thus highlighting a key attribute of these ASLs to span four bases of a codon (Dunham et al., 2007). Another distinguishing feature is that ASLCCCG and ASLACCC do not exhibit the classic U-turn characteristic of canonical ASLs, which is formed by cross-strand interaction involving U33 (Kim et al., 1974; Robertus et al., 1974). Rather, U33 or the additional base at position 33.5 are oriented such that they are unable to participate in stabilizing interactions with the other side of the anticodon loop; thus, the RNA backbone consequently adopts a wider conformation that can span four nucleotides (Dunham et al., 2007). For the ASLSufJ, which contains an expanded anticodon loop due to a nucleotide insertion at position 31.5, the three-nucleotide anticodon forms canonical Watson-Crick base-pairing with the first three bases of the +1 suppressible codon (Fagan et al., 2014). Despite the potential to form Watson-Crick base-pairing between fourth nucleotide in the A-site codon and U33 of ASLSufJ , this interaction is not observed (Fagan et al., 2014). Surprisingly, the trajectory of the anticodon loop nucleotides 33-37 is essentially indistinguishable from a canonical ASL, and structural deviation is observed in the conformation of the phosphate backbone of the anticodon stem to accommodate the presence of the 31.5 base insertion (Fagan et al., 2014). For ASLSufA6, which contains a base insertion at position 37.5, nonstandard base-pairing (a C34·C6 mismatch) occurs at the wobble position during decoding of a +1 suppressible codon (Maehigashi et al., 2014). The presence of the additional nucleotide within the anticodon loop promotes widening of the minor groove of the anticodon stem-loop adjacent to the insertion, between nucleotides 31-33 of the tRNA (Maehigashi et al., 2014). Furthermore, because base 37.5 occupies the position of A38, its presence disrupts a key U32-A38 interaction which is important for stability of the anticodon stem-loop (Maehigashi et al., 2014). Interestingly, electron density corresponding to U32 is absent from the interactions of  ASLSufA6 with its +1 suppressible codons, suggesting that conformational flexibility of U32 may be important for its 25  frameshifting mechanism (Maehigashi et al., 2014). Thus, the structural models of these suppressor tRNA ASLs allude to the importance of  tRNA plasticity in facilitating +1 frameshifting.   Given these examples, it is apparent that recoding is a dynamic process that is ultimately achieved through the influences of cis-acting elements that act on an elongating ribosome, and invariably involves tRNAs. Because the rules that govern how the genetic code is deciphered are transiently altered during decoding, it represents another mechanism by which the diversity of gene expression is achieved.  1.5 Dicistroviridae family of viruses  The Dicistroviridae belong to a family of non-enveloped, monopartite RNA viruses (Bonning and Miller, 2010). The viral family belongs to the order Picornavirales due to similarities in genome sequence and virion structure with Picornaviridae, and contains the Cripavirus and Aparavirus genera which are distinguished based on phylogenetic distance and structure of the intergenic region internal ribosome entry site.  Dicistroviruses are pathogenic to arthropods that have agricultural and medical relevance. For example, the Taura syndrome virus has been attributed as the causative agent of Taura syndrome in panaeid shrimp, which has devastated and caused substantial economic losses in the shrimp industry in Latin America and the United States (Bonami et al., 1997). Additionally, Triatoma virus is pathogenic to the vector of the protozoan parasite, Trypanosoma cruzi, which causes Chagas disease (Muscio et al., 1987). Furthermore, studies have implicated Israeli acute paralysis virus in the colony collapse disorder of honey bees characterized by the loss of worker bees (Cox-Foster et al., 2007; Maori et al., 2007), although there are recent studies suggesting the impact of other emerging factors, including the adverse effects of pesticides, the Varroa destructor parasitic mite, and other viral pathogens (Evans and Schwarz, 2011; Kielmanowicz et al., 2015; Nazzi et al., 2012). Honey bees play an essential role in the pollination of specific crops and their inexplicable decline has been detrimental to the agricultural industry. Despite the importance of honey bees in the agricultural and economic sectors, their virus-host interactions are 26  poorly characterized. An understanding of how viral pathogens and other factors may contribute to disease will lead to effective strategies for disease management and in promoting honey bee health. 1.5.1 Genome organization   The dicistrovirus genome is comprised of a positive-sense, single-stranded RNA which bears a genome-linked viral protein (VPg) covalently linked to the 5'-terminus and a poly(A) tail at the 3'-end (Figure 1.6) (reviewed in (Bonning and Miller, 2010)). The term 'Dicistroviridae' is derived from the unique, bicistronic arrangement of the genome. Whereas other members of the order Picornavirales contain a monocistronic RNA which encodes a polyprotein that is post-translationally processed into both non-structural and structural viral proteins, the genome of dicistroviruses encodes two open reading frames that are differentially regulated by distinct internal ribosome entry sites (IRESs) (Wilson et al., 2000b). Both cistrons are translated as polyprotein precursors that undergo subsequent processing by the virally-encoded protease to yield mature proteins. The upstream cistron, which is under the regulation of the 5'-untranslated region (5'-UTR) IRES, encodes viral non-structural proteins, including a suppressor of RNA-mediated silencing, helicase, 3C-like protease, and RNA-dependent RNA polymerase. The downstream cistron encodes the viral capsid proteins, VP1-4, which is regulated translationally by the intergenic region (IGR) IRES. This unusual bicistronic arrangement of the dicistrovirus genome allows independent and temporal control in the expression of the two open reading frames (Khong et al., 2016), whereby the structural proteins are expressed in supramolar excess over non-structural proteins during infection (Garrey et al., 2010; Moore et al., 1980; Wilson et al., 2000b).  The mechanism of translational initiation on dicistrovirus 5'-UTR IRESs remains largely unexplored due to the lack of sequence and structural conservation. Limited studies using the Rhopalosiphum padi (RhPV) virus 5'-UTR IRES have demonstrated that it exhibits an absolute dependence on eIF2, eIF3, eIF1 and its activity is stimulated by eIF1A, eIF4A, and eIF4G (or its carboxy-terminal fragment) (Terenin et al., 2005). Like putative cellular IRESs, the boundaries of the RhPV 5'- 27      Figure 1.6 Genome organization of Dicistroviridae. The Dicistroviridae genome is comprised of a positive, single-stranded RNA molecule that bears a 5' genome-linked viral protein (VPg) and a 3' poly(A) tail. The genome consists of two open reading frames that are differentially regulated by two distinct internal ribosome entry sites. The upstream cistron encodes nonstructural proteins including suppressor of RNA-mediated silencing (SS), superfamily 3 helicase (hel), VPg, 3A-like protein, chymotrypsin-like cysteine protease (pro), and RNA-dependent RNA polymerase (RdRp). The downstream cistron encodes structural proteins, VP1 to VP4. The upstream and downstream open reading frames are independently regulated by the 5'-untranslated region IRES and intergenic region IRES, respectively. Adapted with permission from (Bonning and Miller, 2010).   28  UTR IRES cannot be unambiguously defined, as IRES activity is only nominally affected with substantial 5'- and 3'-deletions; therefore, it has been proposed that this IRES likely functions by nonspecific binding of key initiation factors proximally to the initiation codon (Terenin et al., 2005). This may account for its cross-kingdom activity in mammalian, insect, and plant translation extracts (Royall et al., 2004; Woolaway et al., 2001). In contrast, the related Cricket paralysis virus (CrPV) and Plautia stali intestine virus (PSIV) 5'-UTR IRESs do not support cross-kingdom activity, thus suggesting dependence on host-specific trans-acting factors for translation (Shibuya and Nakashima, 2006; Wilson et al., 2000b). Recently, Halastavi arva virus (HaIV), an RNA virus containing a dicistronic genome, was shown to employ a mechanism of initiation similar to the RhPV 5' IRES that is primarily dependent on extensive single-stranded regions flanking the start codon (Abaeva et al., 2016). The IRES element within its 5'-UTR promotes direct recruitment of the 43S complex to, or immediately downstream of, the authentic start codon, which subsequently undergoes retrograde scanning to locate the initiation site (Abaeva et al., 2016). The initiation factors eIF2 and eIF3 are absolutely essential, but translation initiation is significantly enhanced in the presence of eIF1 and eIF1A (Abaeva et al., 2016). Because both the RhPV and HaIV 5'-UTR IRESs utilize unstructured sequences proximal to the initiation site to recruit the translational apparatus, it is suggested that this mode of translation initiation may be widespread and conserved in some dicistrovirus 5'-UTR IRESs.  In contrast to the dicistrovirus 5'-UTR IRESs, the translational mechanism of the intergenic region (IGR) IRES has been studied extensively. Structural and biochemical studies reveal that the IGR IRES is comprised of three pseudoknots which fold independently into two functional domains (reviewed in (Jan, 2006; Kieft, 2008; Nakashima and Uchiumi, 2009)). Despite sequence diversity, the secondary structure of the IGR IRES is well conserved across the viral family, and is central to its ability to directly recruit and position the ribosome in the absence of all canonical initiation factors (Nakashima and Uchiumi, 2009). Structural and mechanistic insights into the pathway of IGR IRES-mediated translation initiation will be discussed in detail in Section 1.5.3. 29  1.5.2 Viral-host interactions  How dicistroviruses subvert the host antiviral response remains to be elucidated. The current understanding of fundamental virus-host interactions in invertebrates is still limited, and given the recent epidemic due to Zika virus, an improved understanding of insect biology will be pertinent in dissecting the molecular mechanisms of disease. Dicistrovirus infection thus serves as an excellent model to elucidate virus-host interactions and conserved innate immune response in invertebrates.    Viruses are obligate intracellular pathogens that rely on the host mechanisms to mediate viral protein synthesis and to replicate their genomes. Because viral genomes are compact, they do not encode the necessary components of the translational machinery. Thus, viruses must usurp available cellular resources and divert them toward viral translation. In order to accomplish this, viruses must contend with host messenger RNAs that are being translated through the canonical initiation pathway and evade surveillance by the host innate response. The intricacies of the response that is elicited during dicistrovirus infection have not been examined in detail, and work in elucidating viral-host interactions has only began recently (Garrey et al., 2010; Khong et al., 2016).     Because the majority of cellular transcripts is translated by the cap-dependent pathway, viruses commonly target key canonical initiation factors to attenuate global protein synthesis. This strategy facilitates dampening of the host immune response in infected cells and effectively increases the pool of available ribosomes for viral translation. CrPV infection in Drosophila S2 cells induces rapid host translational shutoff between two to three hours post-infection, and a concomitant increase in the synthesis of viral non-structural and structural proteins (Garrey et al., 2010; Khong et al., 2016). Inhibition of protein synthesis is accomplished by multiple mechanisms dependent on active viral translation and/or replication. Early in infection, formation of the eIF4F complex is impaired through dissociation of eIF4G and eIF4E, which is correlated with host translational shutoff (Garrey et al., 2010). Additionally, eIF2α is phosphorylated later in infection, although it is neither a prerequisite for inhibition 30  of host translation, nor does it perturb the synthesis of viral proteins (Garrey et al., 2010; Khong et al., 2016). Importantly, the activity of the IGR IRES is refractory to or may be stimulated by eIF2α phosphorylation (Fernandez et al., 2002; Thompson et al., 2001; Wang and Jan, 2014; Wilson et al., 2000a). Conceivably, inactivation of eIF2 may promote preferential IRES-mediated translation. During infection, the expression of viral non-structural and structural proteins is temporally regulated. Radioactive pulse-labeling and ribosome profiling experiments suggest that the activity of the 5'-UTR IRES predominates early in infection up to approximately 3 hours post-infection, after which the activity of the IGR IRES is significantly enhanced (Khong et al., 2016). This distinct translation profile remains unaltered even upon premature induction of host translational shutoff by drug treatment (Khong et al., 2016). Thus, precise temporal control may serve as a streamlined mechanism by which optimal expression of non-structural proteins is achieved, prior to the synthesis of structural proteins, for efficient viral packing.   CrPV infection induces rapid translational repression and eIF2α phosphorylation (Garrey et al., 2010; Khong et al., 2016), conditions that are amenable to stress granule formation. In response to specific cellular stresses, polyribosomes are disassembled to facilitate the recruitment of stalled initiation complexes into cytosolic aggregates, called stress granules (SGs), that enable remodeling and/or redistribution of ribonucleoproteins (reviewed in (Beckham and Parker, 2008; Buchan and Parker, 2009)). Classical SGs are induced in Drosophila cells by diverse stresses including heat shock, arsenite, and pateamine A treatment (Farny et al., 2009; Khong and Jan, 2011). Surprisingly, despite robust host translational shutoff and eIF2α phosphorylation, SG formation is inhibited at later time points during CrPV infection, even upon treatment with potent SG inducers (Khong and Jan, 2011). Instead, novel poly(A)+ RNA granules that are distinct from viral replicative intermediates were observed (Khong and Jan, 2011). Interestingly, transient transfection of the CrPV 3C protease in the absence of viral infection results in its sequestration into SGs (Khong and Jan, 2011). Presumably, inhibition of SG formation may 31  be necessary during infection to allow the 3C protease to remain available for processing of the viral polyprotein or cleavage of alternative substrates (Khong and Jan, 2011).  1.5.3 Dicistroviridae intergenic region internal ribosome entry site  The activity and unique properties of the IGR IRES enable the dicistrovirus genome to be preferentially translated during infection despite inhibition of global protein synthesis. As described previously, the IGR IRES obviates the need for all canonical translation initiation factors and can mediate direct recruitment of the 40S and 60S ribosomal subunits for formation of elongation-competent 80S ribosomes. Although the primary sequence is variable across the viral family, with the exception of select residues in single-stranded loop regions that mediate specific contacts with ribosomal components, the overall two-domain conformation of the IGR IRES is conserved (Nakashima and Uchiumi, 2009). Based on the secondary structure, the IGR IRESs are classified into two subtypes, designated as Type I and Type II, which are best exemplified by the CrPV and Israeli acute paralysis virus (IAPV) IRESs, respectively. The primary distinguishing features between the two subtypes include a larger loop L1.1 region, and an additional stem-loop (SLIII) in the tRNA-like domain for Type II IRESs (see Figures 1.7A and 3.1). Despite these structural differences, Type I and II IRESs are thought to adopt a similar fold, and the modular nature of the IRES enables the two domains to be interchangeable between the subtypes to generate functional, chimeric IRESs (Hertz and Thompson, 2011; Jang and Jan, 2010).   Structural and biochemical studies have demonstrated that the IGR IRES adopts an overlapping, triple-pseudoknot structure consisting of pseudoknots (PKs) I, II, and III, which together constitute two independently folded domains (Figure 1.7) (Jan, 2006; Kieft, 2008; Nakashima and Uchiumi, 2009). PKII/PKIII adopt a compact, solvent-inaccessible core which is primarily responsible for ribosome binding (Costantino and Kieft, 2005; Jan and Sarnow, 2002; Nishiyama et al., 2003). Within this domain, stem-loops (SL) IV and V protrude and establish critical contacts with the 40S subunit via ribosomal proteins (rp) S5 and S25 (Nishiyama et al., 2007; Pfingsten et al., 2006; Schuler et al., 2006). Ribosomes 32  deficient in rpS25, which localizes to the head domain of the 40S ribosomal subunit, exhibit significantly diminished binding of the CrPV and PSIV IRESs (Landry et al., 2009; Muhs et al., 2011). The apical loop sequences of SLIV and SLV are nearly invariant across the IGR IRESs, highlighting the importance of these interactions for IRES-mediated translation initiation. The conserved L1.1 region facilitates 80S formation via interaction with the L1 stalk of the 60S subunit (Pfingsten et al., 2006; Spahn et al., 2004a). Bulk substitutions or mutation of specific nucleotides within L1.1 render the IRES less competent in assembling 80S ribosomes despite robust binary IRES-40S formation (Jang and Jan, 2010; Pfingsten et al., 2006).   PKI constitutes a tRNA-like domain that is primarily responsible for establishing the translational reading frame (Costantino et al., 2008). High resolution structural analysis of the CrPV IGR IRES revealed that the PKI mimics an authentic anticodon:codon interaction, where the topology of the sugar-phosphate backbone and the positioning of the residues that constitute PKI are essentially identical to an anticodon loop (Costantino et al., 2008) (Figure 1.7). PKI deviates from the canonical anticodon:codon pairing in being comprised of five base-pairing interactions, which together translocate into the P site to permit delivery of the first aminoacyl-tRNA. Furthermore, the presence of an additional base, A6191, in the PKI domain, which has no corresponding counterpart in the initiator-tRNA, consequently introduces a shift in the adjacent nucleotide G6192 such that its position is offset from that of the analogous base in the tRNA anticodon loop (Costantino et al., 2008).  Strikingly, the PKI domain resembles a P/E hybrid state tRNA site, which is a conformational intermediate that is only achieved during translation elongation; therefore, precise tRNA-mRNA mimicry likely enables the IGR IRES to activate the translational apparatus and prime the ribosome into an elongation mode of translation (Costantino et al., 2008). Consistent with this, low-resolution cryo-EM studies reveal that IGR IRES binding induces various conformational changes within the ribosome (Schuler et al., 2006; Spahn et al., 2004a). IRES binding promotes a rotation of the head domain of the 40S subunit, and the formation of latch interactions between helices 18 and 34 of 18S rRNA near the entrance of the mRNA channel (Schuler et al., 2006; 33  Spahn et al., 2004a). Interestingly, similar bridging interactions are observed upon binding of the unrelated HCV IRES to the solvent-exposed face of the ribosome, which suggests that this may be a feature intrinsic to translation which may facilitate movement of the transcript through the mRNA cleft (Spahn et al., 2001).   1.5.3.1 Mechanism of IGR IRES-mediated translation initiation  Previous low-resolution cryo-EM reconstructions have demonstrated that the IGR IRES is localized in the intersubunit space of the ribosome and traverses the ribosomal A-, P- and E-sites where tRNAs bind (Schuler et al., 2006; Spahn et al., 2004a). These structures provided the basis of a preliminary model of IRES-mediated translation, wherein the PKI domain may dock into the ribosomal P site upon initial binding to the ribosome to allow the incoming aminoacyl-tRNA to access the non-AUG initiation codon in the A site (Wilson et al., 2000a). Recently, advances in cryo-EM technologies have enabled the acquisition of high-resolution structural models that have yielded unexpected mechanistic insights into IRES-mediated translation (Fernandez et al., 2014; Koh et al., 2014; Muhs et al., 2015). Most prominently, the structural data show unambiguously that the PKI domain is positioned in the A site of the ribosome (Fernandez et al., 2014; Koh et al., 2014) (Figure 1.8). Additionally, these structures reveal that PKI closely mimics the anticodon stem-loop of tRNA.  Ribosome recruitment to the IGR IRES occurs predominantly via a sequential pathway, whereby the 40S subunit is assembled prior to 60S subunit joining. At a lower frequency, however, ribosomal subunits can be simultaneously recruited to the IRES RNA in vitro (Petrov et al., 2016). The dynamics of the 40S head domain may facilitate positioning of the initiator Met-tRNAi in the P site in canonical translation (Aylett et al., 2015; Llacer et al., 2015). Interestingly, IRES binding restricts the conformational flexibility of the 40S head through insertion of SLIV and SLV into the cleft between the 40S head and body domains (Murray et al., 2016) (Figure 1.8). The PKI domain establishes ribosomal contacts in the decoding center that mimic those during canonical decoding, including the interaction of  34   Figure 1.7 Dicistroviridae intergenic region internal ribosome entry site.  (A) Sequence and secondary structure of the Cricket paralysis virus (CrPV) IGR IRES. Stem-loops (SL) IV and V interact with the 40S subunit via ribosomal proteins uS7 and eS25. Loop L1.1 interacts with the L1 stalk of the 60S subunit. Notable structural elements are boxed in the corresponding colors as depicted in the crystal structure in (B). The PKI domain structurally mimics an authentic anticodon:codon interaction (boxed in green) to establish the translational reading frame and initiate translation from the A site at a non-AUG initiation codon. Specific nucleotides within this region are depicted in the corresponding colors as the structural shown in (C). (B) Crystal structure of the ribosome binding domain from the Plautia stali intestine virus IRES. (C) Comparison of the CrPV PKI anticodon:codon-like interaction and an authentic P-site tRNA-mRNA interaction. Analogous bases in both structures are highlighted in the same color. (D) Cryo-EM reconstructions of the vacant human 40S ribosomal subunit (left) and the CrPV IGR IRES-bound 40S complex (right) at 25.3 Å and 20.3 Å, respectively. The IGR IRES binds to the intersubunit space and induces conformational changes in the 40S subunit (indicated by asterisk). Reproduced with permission from (Au and Jan, 2014). 35  the conserved 18S ribosomal RNA (rRNA) bases A1756 and A1755 (1493/92 in E. coli) with the minor groove of the IRES anticodon:codon mimic (Fernandez et al., 2014; Koh et al., 2014; Muhs et al., 2015). However, interactions that are exclusive to IRES-mediated translation initiation have also been reported. For example, while C1273 (or C1054 in E. coli) of 18S rRNA interacts with the wobble position of the anticodon during canonical decoding, a rotation about its glycosidic bond enables it to engage in stacking interactions with the CrPV RNA in IRES-mediated translation (Murray et al., 2016). Consequently, C1273 can base-pair directly with the first nucleotide in the initiation codon of the IRES (Abeyrathne et al., 2016; Murray et al., 2016). It is interesting to note that with the exception of the PSIV IRES, all IGR IRESs initiate with a guanine nucleotide, which may suggest functional relevance of this observed interaction with 18S rRNA.  Following 60S subunit joining, the IRES/80S complex can fluctuate between the ratcheted and classical pre-translocation states, which are similar to the rotational states of the ribosome observed during translocation (Fernandez et al., 2014; Koh et al., 2014; Murray et al., 2016). These two states are differentiated by the counter-clockwise rotation of the small subunit relative to the large ribosomal subunit in the ratcheted state, 40S head domain swivelling and movement of the L1 stalk. Loop L1.1 of the IRES interacts with the L1 stalk to couple its movement with the rotational state of the ribosome (Koh et al., 2014; Murray et al., 2016). Because the IRES maintains interactions with these dynamic ribosomal components with the PKI domain anchored in the A site, the IRES adopts variable conformations that are dependent on the rotational state of the ribosome (Fernandez et al., 2014). The IRES-ribosome complex can be trapped in an intermediate translocation state upon incubation with eEF2 and non-hydrolyzable GTP or in the presence of the GTP and the translation inhibitor sordarin (Abeyrathne et al., 2016; Murray et al., 2016). The pre-translocation state is characterized by an additional small subunit rotation and an outward displacement of the L1 stalk, which moves in an inward (opposite) direction during normal translation to stabilize tRNA in the P/E hybrid state (Fernandez et al., 2014; Koh et al., 2014; Muhs et al., 2015). Interaction of PKI with domain IV of eEF2 displaces the anticodon stem-loop-like element of PKI  36   Figure 1.8 Pathway of IGR IRES-mediated translation initiation. The IGR IRES interacts with the 40S ribosomal subunit via SL IV and V. This interaction restricts the conformational flexibility of the 40S head domain. Following 60S subunit joining, the IRES/80S complex fluctuates between canonical and rotated states, where PKI is anchored in the A site ('pre-translocation states'). The binding of eEF2 to the rotated state induces an additional ~3° rotation of the 40S and displacement of the L1 stalk to a wider conformation. Interaction of Domain IV of eEF2 with PKI stabilizes it in an intermediate translocation state, reminiscent of an ap/P state. During translocation,  GTP hydrolysis causes eEF2 and the ribosome to undergo conformational changes that promote the translocation of PKI to the ribosomal P site, during which the initial contacts of SL IV and V with the 40S subunit are disrupted ('post-translocation state'). Reproduced with permission from (Murray et al., 2016). 37  to an intermediate position between the A and P sites, reminiscent of  the ap/P state of tRNA (Abeyrathne et al., 2016; Murray et al., 2016). As PKI transitions from the A to P sites, interactions between region L1.1 with the L1 stalk and SLIV/SLV with uS7, uS11 and eS25 are maintained (Abeyrathne et al., 2016). Global conformational rearrangements are also observed for the IRES, as it varies from extended (initiation state), to compact (pre-translocation intermediates) to extended conformations (post-translocation state), likened to an inchworm movement through the ribosome (Abeyrathne et al., 2016). The diphthamide modification of eEF2 residue H699 packs against the ribose of G6189 of CrPV IRES, which interferes with the interaction of the decoding rRNA bases A1755 and A1756 with the PKI helix in the A site (Abeyrathne et al., 2016; Murray et al., 2016). This additional stabilizing interaction contributed by the diphthamide moiety likely facilitates the translocation of PKI from the A site to the P site (Murray et al., 2016). The role of diphthamide in IRES-mediated translation is further supported by in vitro experiments which demonstrated that IRES-directed tripeptide synthesis was significantly impaired using eEF2 that lacked diphthamide, while a negligible effect on tripeptide synthesis was observed for the canonical initiation pathway (Murray et al., 2016); thus, diphthamide modification may be important in mediating the initial pseudotranslocation steps prior to the first peptide bond formation.   Delivery of the first aminoacyl-tRNA can only occur following pseudo-translocation of PKI from the A site to the P site (Fernandez et al., 2014; Koh et al., 2014). Previous toeprinting experiments have demonstrated that, in the absence of both eEF1A and cognate aminoacyl-tRNA, the toeprint position does not change upon incubation of IRES-ribosome complexes with eEF2 (Jan et al., 2003; Pestova and Hellen, 2003). While this is seemingly contradictory to the current paradigm, it is suggested that a translocated PKI in the P site is susceptible to spontaneous back-translocation unless stabilized by an A-site ligand (Fernandez et al., 2014). Indeed, the post-translocated conformation of the IRES can be captured when eukaryotic release factor 1 (eRF1) is bound to a modified IRES/ribosome complex which bears a stop codon in the first position (Muhs et al., 2015). eRF1 binds to the termination codon in an eEF2-dependent manner (Jan et al., 2003) and acts as an A site ligand to trap the PKI in the post-translocated state (Muhs 38  et al., 2015). During pseudo-translocation, hydrolysis of eEF2-bound GTP disrupts the interactions of SLIV and SLV with the small ribosomal subunit, causing them to become solvent-accessible, while interaction with the L1 stalk is maintained (Abeyrathne et al., 2016; Muhs et al., 2015; Murray et al., 2016). The loss of interaction with the small ribosomal subunit facilitates the movement of PKI into the P site, which is also accompanied by lateral displacement of the ribosome binding domain toward the E site (Muhs et al., 2015). This conformational rearrangement renders the A site vacant for the first incoming aminoacyl-tRNA, and primes the IRES for subsequent elongation steps. 1.5.3.2 IRES-mediated alternate reading frame selection  Bioinformatic analyses have identified enhanced coding potential in the honey bee dicistroviruses Israeli acute paralysis virus, Acute bee paralysis virus, Kashmir bee virus and the fire ant virus Solenopsis invicta virus-1 via an overlapping gene (Firth et al., 2009; Sabath et al., 2009) (Figure 1.9). The alternate gene, ORFx, is encoded in the +1 translational reading frame within the 5' proximal region of the cistron encoding viral structural proteins (Firth et al., 2009). Expression of ORFx was verified in vitro using reporter constructs in Spodoptera frugiperda (Sf21) transcription-translation coupled extracts (Ren et al., 2012). +1 frame translation occurs at approximately 20% frequency of 0 frame translation, and initiates via a U:G wobble base-pair adjacent to the IRES translational start site (Ren et al., 2012). Interestingly, mutagenesis studies have identified point mutations within the PKI anticodon stem-loop-like element that result in exclusive 0 or +1 frame translation, suggesting that additional IRES elements may contribute to reading frame selection (Ren et al., 2014). Because ORFx translation occurs in an IRES-dependent manner, its translation represents a novel mode of alternate reading frame selection directed by an IRES, unlike conventional ribosomal frameshift mechanisms (Ren et al., 2012). While the function of ORFx is not currently known, mass spectrometry has confirmed that it is expressed in virus-infected honey bees (Ren et al., 2012). Additionally, the maintenance of the ORFx sequence under selective pressure suggests that it may have a relevant biological function during the viral life cycle. Recent kinetic studies have yielded detailed mechanistic insights into the initial translocation events  39    Figure 1.9 Identification of a novel overlapping reading frame in a subset of dicistroviruses. Alignment of 16 bee dicistrovirus CDS2 sequences, with the presence of stop codons in all three translational reading frames depicted by triangles. In the +1 frame, the conserved absence of stop codons in the 5'-proximal region suggests the location of the putative ORFx. Adapted with permission from (Firth et al., 2009).    40  during IRES-mediated translation (Zhang et al., 2016). These studies reveal that ribosome occupancy by the IRES exerts retarding effects on the first two translocation events (Zhang et al., 2016). Following translocation of the tripeptidyl-tRNA, subsequent translocation steps occur at increased rates (Zhang et al., 2016). While the implications of the retarding effects are not understood, it is possible that this phenomenon may facilitate and/or contribute to selection of the reading frame .   1.6 Thesis investigation   The IGR IRES utilizes a streamlined mechanism to recruit and bind ribosomes independently of initiation factor activities. Much of our current understanding of IRES-mediated translation has been derived from structural and biochemical studies that reveal precise tRNA-mimicry by the IRES to co-opt the translational apparatus. Recent high-resolution cryo-EM models have also provided structural insights into how the IRES exploits the canonical elongation cycle to drive pseudo-translocation, thus providing a more holistic view of the conformational rearrangements that underlie IRES-mediated translation. However, despite these significant advances in understanding, the mechanism of reading frame selection during IRES-mediated translation initiation remains an outstanding question. Because the IGR IRES extensively mimics a tRNA, we hypothesize that specific IRES-ribosome contacts facilitate reading frame selection. Here, we adopt various biochemical assays to rigorously address the contribution of specific IRES structural elements to reading frame selection. In Chapter 2, we explored how constituents of the PKI domain, including the PKI helical stem, the variable loop region (VLR), and the anticodon-codon-like base-pairing, contribute to reading frame selection. By adopting a mutagenesis approach, we demonstrate that the dynamic nature of the VLR is important for IRES-mediated translation. We also provide biochemical evidence that the PKI domain mimics a codon:anticodon interaction by generation of a functional - albeit weak - chimera through replacement of the IRES anticodon mimic with the anticodon stem-loop of a true tRNA. In Chapter 3, we explored the functional role of stem-loop III in Israeli acute paralysis virus IRES-mediated reading frame selection. We utilized a bicistronic reporter assay to 41  demonstrate that SLIII formation is critical for IRES-mediated 0 frame translation, but not +1 frame translation. In collaboration with Dr. Samuel Butcher (University of Wisconsin-Madison), we determined the structure of the IAPV PKI domain by nuclear magnetic resonance/ small angle X-ray scattering (NMR/SAXS) hybrid approach and demonstrate that it adopts the L-shape conformation characteristic of an authentic tRNA. In Chapter 4, we investigated the role of SLVI during virus infection. Because the presence of SLVI is conserved in a subset of dicistroviruses infectious to honey bees, we hypothesize that it has an intrinsic role in the viral life cycle. Due to the lack of a bona fide infectious clone of a honey bee dicistroviruses, we derived a chimeric virus from the full-length molecular clone of the related Cricket paralysis virus. Using this chimera, we demonstrate that formation of SLVI is critical for optimal IGR IRES-mediated synthesis of the viral structural proteins, and that disruption of SLVI decreases viral yield. Comprehensive biochemical analysis of different IRES structural elements demonstrate that they all contribute to optimal IRES function and activity.      42  CHAPTER 2: THE INTEGRITY OF THE IGR IRES PSEUDOKNOT DOMAIN IS IMPORTANT FOR IRES-MEDIATED TRANSLATION INITIATION 2.1  Introduction  Eukaryotic translation initiation can proceed via different pathways. While the majority of cellular mRNAs undergo cap-dependent translation initiation, some viral RNAs rely on IRES elements to hijack the host translational machinery. During virus infection or other cellular stresses, the activities of specific translation initiation factors may become compromised. Because IRES elements can recruit and position ribosomes using a substantially reduced complement of the canonical initiation factors, they remain refractory to many forms of translational inhibition and can direct efficient synthesis of viral proteins. One classical example occurs in the hepatitis C virus (HCV), which harbours a Group 2 IRES that directly binds the 40S subunit and requires only eIF3 and the ternary complex of eIF2-GTP-Met-tRNAi for translation initiation (Pestova et al., 1998). The functional core of the HCV IRES consists of a four-way helical junction that adopts a double-pseudoknot fold (Berry et al., 2010; Berry et al., 2011). This pseudoknot domain contributes directly to the appropriate positioning of the initiator Met-tRNAi at the AUG start codon of the HCV IRES (Berry et al., 2010; Berry et al., 2011; Wang et al., 1995).    The intergenic region (IGR) IRES of the Dicistroviridae family also contains a pseudoknot structure (PKI) to direct ribosome positioning at the initiation codon. However, unlike the HCV IRES, the IGR IRES functionally supplants initiation factors and acts as an all-RNA equivalent that manipulates and hijacks the ribosome for viral protein synthesis without the use of initiator Met-tRNAi (Jan et al., 2003; Jan and Sarnow, 2002; Pestova and Hellen, 2003; Sasaki and Nakashima, 1999, 2000; Wilson et al., 2000a). The PKI tRNA mimicry domain positions the ribosome on the IRES such that translation initiates within a specific reading frame and at a specific non-AUG codon from the ribosomal A site (Costantino et al., 2008; Fernandez et al., 2014; Jan et al., 2003; Jan and Sarnow, 2002; Koh et al., 2014; Pestova and Hellen, 2003; Sasaki and Nakashima, 1999, 2000; Wilson et al., 2000a). Several biochemical approaches 43  have revealed that the IGR IRES undergoes conformational rearrangements in the progression from the unbound state, to the 40S subunit- and 80S-bound states (Costantino et al., 2008; Jan and Sarnow, 2002; Nishiyama et al., 2003; Pfingsten et al., 2010; Pfingsten et al., 2006). Specifically, biochemical and enzymatic probing studies have demonstrated that select nucleotides within PKI are accessible to reagents that target both single-stranded and helical regions, thus suggesting that this domain of the IRES may be structurally dynamic (Jan and Sarnow, 2002; Nishiyama et al., 2003). Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis of PKI has also revealed that numerous local conformational changes occur and that specific nucleotides become hypermodified in the 80S- compared to the 40S-bound state (Costantino et al., 2008). Currently, it is not understood how and at which step these conformational changes contribute to IRES-mediated translation. To gain insight into this process, we performed a comprehensive mutagenesis study targeting three constituents of the PKI tRNA mimicry domain, including the PKI helical stem, the codon:anticodon-like base-pairing interactions, and the variable loop region  (VLR) and assessed their functional contribution to ribosome positioning which represents a key step of IRES-mediated translation initiation. Alterations to the PKI helical stem by introduction of nucleotide bulges or base-pair deletions generally reduced IRES translation, suggesting a functional importance of this region to IRES translation. Furthermore, the defect in translation and ribosome positioning observed with the VLR insertion/deletion mutations suggests that there is a stringent requirement in the length of the VLR for IRES activity and that this region may be involved in optimal docking of the IRES within the ribosome to drive translation. Here, we have elucidated the molecular parameters of the PKI tRNA mimicry domain that contribute to IGR IRES-mediated translation and reading frame selection.  2.2 Materials and methods 2.2.1 DNA constructs The bicistronic luciferase plasmid containing the CrPV IGR IRES has been previously described (Jan and Sarnow, 2002). Mutant IRES constructs were generated using PCR-based mutagenesis and verified by 44  sequencing (see Appendix A). PKI chimeras encompassing sequences from prolyl-tRNA were designed using the sequence of human prolyl-tRNA (Chang et al., 1986).  2.2.2 In vitro transcription and translation The bicistronic DNA construct containing the wild-type or mutant IRES was linearized using XbaI. RNAs were transcribed in vitro using T7 RNA polymerase and subsequently purified using an RNeasy Mini Kit (Qiagen). The purity and integrity of the RNAs were confirmed by gel electrophoresis. For in vitro translation assays, uncapped RNAs (15 ng/µL) were pre-folded by heating in water at 65°C for 3 minutes, followed by addition of Buffer E (20 mM Tris-Cl [pH 7.5], 100 mM potassium acetate (KOAc) [pH 7.0], 2.5 mM magnesium acetate (MgOAc), 0.25 mM spermidine, 2.0 mM dithiothreitol (DTT)) and subsequent incubation at 30°C. The pre-folded RNAs were incubated in a rabbit reticulocyte lysate (RRL) translation extract (Promega) supplemented with additional KOAc to a final concentration of 154 mM and incubated for 1 hour at 30°C. Luciferase reporter expression was monitored by [35S] incorporation (EasyTag Express Protein Labeling Mix, PerkinElmer). The reactions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were dried and subjected to Phosphorimager analysis (Typhoon, GE Healthcare).    2.2.3 Purification of 40S and 60S ribosomal subunits 40S and 60S ribosomal subunits were purified from HeLa pellets (National Cell Culture Center), as described previously (Jan and Sarnow, 2002). HeLa cells were lysed in lysis buffer (15 mM Tris-Cl [pH 7.5], 300 mM NaCl, 1% (v/v) Triton X-100, 6 mM MgCl2, 1 mg/mL heparin) and the supernatant was subjected to brief centrifugation to remove cellular debris. The supernatant was applied to a 30% (w/w) sucrose cushion containing 500 mM KCl and centrifuged at 100,000 g to pellet ribosomes. Ribosomes were resuspended in Buffer B (20 mM Tris-Cl [pH 7.5], 6 mM MgOAc, 150 mM KOAc, 6.8% (w/w) sucrose, 2 mM DTT) and subsequently treated with puromycin (2.3 mM final concentration) to dissociate ribosomes from the mRNAs. Potassium chloride was added to a final concentration of 500 mM. The 45  dissociated ribosomes were resolved on a 10-30% (w/w) sucrose gradient where the peaks corresponding to the free 40S and 60S subunits were detected by measuring the absorbance at 260 nm. The corresponding fractions were collected, pooled, and concentrated in Buffer C (20 mM Tris-Cl [pH 7.5], 0.2 mM EDTA, 10 mM potassium chloride, 1 mM magnesium chloride, 6.8% (w/w) sucrose) using Amicon Ultra spin concentrations (Millipore). The concentrations of the ribosomal subunits were determined by spectrophotometry using the conversions 1 A260 nm = 50 nM and 1 A260 nm = 25 nM for the 40S and 60S subunits, respectively.  2.2.4 Toeprinting/ primer extension analysis Toeprinting analysis of ribosomal complexes in RRL was performed as previously described (Wilson et al., 2000a). 400 ng of bicistronic wild-type or mutant IGR IRES RNAs were annealed to primer PrEJ69 (5'-GTAAAAGCAATTGTTCCAGGAACCAG-3') in 40 mM Tris-Cl [pH 7.5] and 0.2 mM EDTA by slow cooling from 65°C to 30°C. Annealed RNAs were added to a RRL (6.5 µL) pre-incubated with either 15 µM edeine or 0.68 mg/ml cycloheximide and containing 20 µM amino acid mix, 8 units of Ribolock (Fermentas) and 154 mM final concentration KOAc [pH 7.5]. The reaction was incubated at 30°C for 20 minutes. Following incubation, ribosome positioning was determined using the whole reaction volume by primer extension/reverse transcription using 10 units of AMV reverse transcriptase (Promega) in the presence of 125 µM of each of deoxythymidine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate,  25 µM deoxyadenosine triphosphate, 0.5 µL of α-[32P] deoxyadenosine triphosphate (PerkinElmer, 3.33 µM, 3000 Ci/mmol), 8 mM MgOAc, 10 units of Ribolock (Fermentas), Buffer E (20 mM Tris-Cl [pH 7.5], 100 mM KCl, 2.5 mM MgOAc, 0.25 mM spermidine, 2 mM DTT) in the final reaction volume of 20 µL. The reverse transcription reaction was incubated at 30°C for 1 hour, after which it was quenched by the addition of Stop Solution (450 mM ammonium acetate (NH4OAc), 0.1% SDS, 1 mM EDTA). Toeprinting/ primer extension analysis using purified 40S and 60S ribosomal subunits was performed in a similar manner using 150 ng bicistronic RNA and 100 nM and 150 nM final concentrations of 40S and 60S, respectively, at 37°C. Following the reverse transcription reaction, the 46  samples were extracted by phenol/chloroform (twice), chloroform alone (once), and ethanol precipitated. The complementary DNAs were analyzed under denaturing conditions of 6% (w/v) polyacrylamide/8 M urea gels, which were dried and subjected to Phosphorimager analysis. For quantitation, toeprint intensities were measured as a fraction of the radioactive counts for Toeprint A over the total radioactive counts within each lane, normalized to that of the wild-type IGR IRES. 2.3 Results 2.3.1 Regions of CrPV PKI can be substituted by sequences from the prolyl-tRNA anticodon stem-loop   Despite some deviations from an authentic anticodon:codon interaction, the striking similarity between the structure of the CrPV PKI and an authentic tRNA-mRNA interaction suggests that the CrPV anticodon-like element may be functionally substituted by sequences from an authentic anticodon stem-loop (Costantino et al., 2008; Zhu et al., 2011). To explore this possibility, chimeric IRESs were generated by interchanging regions of PKI with corresponding elements from the human prolyl-tRNA anticodon stem-loop and assessed for functional IRES activity using the standard bicistronic reporter system. Prolyl-tRNA was selected as a fitting candidate because a proline-encoding triplet occupies the ribosomal P site during CrPV IGR IRES-mediated translation initiation (Wilson et al., 2000a). To determine if the prolyl-tRNA anticodon stem-loop can functionally replace the anticodon mimic of the IRES, a substitution was made such that the number of base-pairs within the helical stem is consistent with the wild-type PKI domain (Figure 2.1, ProtRNA). The effect on IRES-mediated translation was assessed in rabbit reticulocyte lysates (RRL) using in vitro transcribed bicistronic reporter RNAs harboring the wild-type or chimeric IRESs in the intercistronic space (Figure 2.2A, top). Expression of the upstream Renilla luciferase (RLuc) which serves as a normalizing control, and the downstream firefly luciferase (FLuc) reporters are driven by scanning-mediated and IRES-mediated translation initiation, respectively. For the ProtRNA construct, the substitution was sufficient to confer translational activity  47    Figure 2.1. Chimeric IRESs containing prolyl-tRNA and wild-type CrPV PKI regions. Top. Schematic of the authentic prolyl-tRNA and wild-type CrPV IRES. The expected location of the positioning toeprint (Toeprint A) is indicated. U6185 and G6192  are depicted in blue. Bottom. Schematic of chimeric PKI domain IRESs that have been substituted by prolyl-tRNA elements (shaded in grey), or bearing mutations in PKI (black box). Mutated nucleotides are depicted in red.    48  albeit weakly (18% of control), suggesting that sequences of an authentic anticodon stem-loop can impart IRES activity (Figure 2.2, ProtRNA). To determine if the IRES helical stem or the stem-loop can be independently replaced by the analogous regions of the prolyl-tRNA, chimeric IRES constructs containing bulk substitutions within the PKI domain were generated (Figure 2.1). For the ProLoop construct which consists of a composite of the IRES helical stem and prolyl-tRNA stem-loop, 29% of wild-type IRES activity was observed (Figure 2.2, ProLoop). The reciprocal chimera, the IRES of ProLoop2, containing the prolyl-tRNA helical stem and wild-type IRES loop, rendered weaker activity (9% of control; Figure 2.2, ProLoop2). Inspection of the secondary structural models of the Type I IRESs reveals that seven of nine members in the subgroup contain a U and G at the base of the helical stem (U6185/G6192 for CrPV IRES, depicted in blue in Figure 2.1) (Nakashima and Uchiumi, 2009). Crystal structures of the CrPV IRES PKI domain reveal that these bases, although conserved, do not base-pair but are essential for IRES function (Costantino et al., 2008). To determine if U6185/G6192 are important for IRES-mediated translation in the context of the chimeras, a C to U base substitution was introduced into ProLoop2 to generate ProLoop1 (Figure 2.1, ProLoop1, mutation depicted in red). Interestingly, this single nucleotide change was sufficient to enhance IRES activity (from 9% to 45% of wild-type) as compared to that of the ProLoop2 construct, suggesting that the identity of the U and G at the base of the stem is critical for IRES-mediated translation (Figure 2.2, ProLoop1). Consistent with the importance of these nucleotides, mutation of U6185 to A, C, or G alone inhibited IRES activity (Costantino et al., 2008).   The moderate activity exhibited by the chimeric IRESs containing the prolyl-tRNA anticodon loop suggests that a three-base-pair interaction in PKI is sufficient for IRES-mediated translation. To determine if this holds true for the wild-type CrPV IRES, UA6190-1 or UA6212-3 were individually mutated to their Watson-Crick complements to abrogate the additional base-pairs within the tRNA mimicry domain (Figure 2.1, UA6190-1AU and UA6212-3AU). When both mutations were introduced concomitantly to restore base-pairing, IRES activity was rescued to ~70% of wild-type (Figure 2.1 and 2.2A and B, Comp). These results demonstrate that three base-pairs are sufficient to confer IRES  49    Figure 2.2. Translational activities of chimeric and mutant IRESs. (A) Top. Uncapped bicistronic reporter RNAs used in translation and toeprinting/ primer extension assays. The upstream Renilla luciferase (RLuc) and downstream firefly luciferase (FLuc) cistrons are expressed by scanning-mediated and IRES-mediated translation, respectively. Bottom. Translational activities. Uncapped bicistronic reporter RNAs were incubated in RRL in the presence of [35S]-methionine, as described in Section 2.2.2. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments. (B) Summary of the translational activities and toeprint intensities for the chimeric and mutant IRESs. (C) Toeprinting analysis of chimeric IRESs. Bicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in the absence [-] or presence [+] of purified salt-washed HeLa 80S (100 nM), as described in Section 2.2.4. The sequencing reactions for each construct are shown on the left, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the right. 50  translation but that formation of the additional base-pairs may further modulate or fine-tune IRES activity, consistent with a previous report also demonstrating that additional PKI base-pairing is optimal for Israeli acute paralysis virus and Taura syndrome virus IGR IRES activities (Hertz and Thompson, 2011). Altogether, these results functionally confirm and further reinforce the previous structural and biochemical evidence that the PKI domain mimics an anticodon:codon interaction to drive IRES translation.   IRES-mediated translation involves ribosome recruitment, positioning and the subsequent translocation step. Upon initial binding to the ribosome, the IRES PKI domain is positioned into the ribosomal A site (Fernandez et al., 2014; Koh et al., 2014) To determine if the defect in IRES-mediated translation for the chimeric IRESs is due to impaired ribosome positioning, toeprinting/ primer extension analysis was performed using purified, salt-washed HeLa 80S ribosomes. The position of the ribosome on the IRES can be determined by reverse transcription primed by an oligonucleotide which hybridizes ~100 nucleotides downstream of the IGR IRES translational start site. Incubation of the wild-type IGR IRES with purified 80S generated a toeprint at CA6226-7 or 'Toeprinting A', which is indicative of proper ribosome positioning (Figure 2.2C, lane 2). The corresponding cDNA is generated upon termination of the reverse transcription reaction due to contact made with the leading edge of the ribosome. This stoppage occurs at +13-14 nucleotides downstream of the CCU triplet in the ribosomal A site, given that the first C is designated as the +1 position (Jan and Sarnow, 2002). A CC6214-5GG mutation (ΔPKI), which effectively abrogates base-pairing within PKI, eliminated Toeprint A (Figure 2.2C, lane 4). For the ProtRNA and ProLoop2 chimeras which exhibited weak IRES activity, Toeprint A was not observed, suggesting a deficiency in ribosome positioning (Figure 2.2C, lanes 6 and 12). Both ProLoop and ProLoop1 chimeras generated Toeprint A but at reduced intensities compared to the wild-type IRES, consistent with their lower translational efficiencies (Figure 2.2B and 2.2C, lanes 8 and 10). These findings suggest that the reduced translational activities of some chimeric IRESs may be due to defective ribosome binding and/or positioning.  51  2.3.2 A single base-pair deletion is tolerated in the PKI helical stem  Having confirmed that the PKI domain can be functionally supplanted by a prolyl-tRNA anticodon loop, we next systematically investigated the three constituents that make up the PKI tRNA mimicry domain. The PKI helical stem of the CrPV IRES resembles a tRNA anticodon stem-loop. However, nucleotide bulges are prevalent within the helical stems of the IGR IRESs, but do not appear to be conserved from secondary structural predictions (Nakashima and Uchiumi, 2009). The significance and function, if any, of these bulges have not been investigated. Within the CrPV IRES A6182 protrudes from the helical axis in the ribosome-unbound structure to establish crystal contacts (Zhu et al., 2011). In the ribosome-bound state, however, this nucleotide extends inward to maintain coaxial stacking within the PKI helical stem (Zhu et al., 2011). Though it is not conserved, deletion of this nucleotide decreased IRES activity to ~74%, suggesting that this nucleotide bulge has a minor but functional role in IRES-mediated translation (Figure 2.3 and 2.4B, ∆A6182). To determine if other nucleotide bulges can be tolerated, one or two adjacent nucleotides were deleted from the 5' proximal region of the helical stem and the resultant effect on IRES activity was assessed (Figure 2.3, ∆U6176 and ∆UU6176-7). Deletion of U6176 or U6176-7 reduced IRES translation to ~50% and 25% of wild-type, respectively (Figure 2.3 and 2.4B , ∆U6176 and ∆UU6176-7). Next, to assess if complete base-pairs can be excluded from the helical stem, the complementary nucleotides were also deleted (Figure 2.3, ∆U6176/A6200 and UU6176-7/AA6199-200). While deletion of one complete base-pair rendered the IRES moderately active (~50%), removal of two complete base-pairs was sufficient to abrogate IRES activity (Figure 2.3 and 2.4B, ∆U6176/A6200 and UU6176-7/AA6199-200). These results, therefore, demonstrate that the PKI helical stem region can tolerate nucleotide deletions that result in the formation of a new bulge, consistent with the lack of conservation in the bulged nucleotides within the helical stem. However, the position of the bulge may be important for optimal IRES activity, as deletion of A6182 adversely affected IRES-mediated translation (Figure 2.3 and 2.4B). Additionally, these results also demonstrate that a one base-   52    Figure 2.3. The IRES helical stem tolerates bulges and a 1-base-pair deletion. (A) Schematic of the IRES PKI helical stem is shown, with the region bearing mutations highlighted in grey. The respective mutations resulting in a 1-nucleotide bulge (∆U6176), 2-nucleotide bulge (∆UU6176-7), 1-base-pair deletion (∆U6176/A6200) and 2-base-pair deletion (∆UU6176-7/AA6199-200) are shown. (B) Translational activities of mutants bearing alterations in the PKI helical stem. Uncapped bicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [35S]-methionine, as described in Section 2.2.2. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments.     53  pair but not a two base-pair deletion is tolerated in the helical stem,  indicating that a minimum helical stem length must be maintained for efficient IRES-mediated translation (Figure 2.3 and 2.4B).   To determine if mutations in the helical stem affected ribosome positioning, toeprinting analysis was performed using purified HeLa 80S in parallel with the wild-type IRES and a mutant deficient in PKI base-pairing (Figure 2.4A). Deletion of A6182, U6176 or U6176/A6200 likely impeded optimal positioning of PKI in the A site, and resulted in reduced Toeprint A intensities (Figure 2.4A, lanes 5-10 and 2.4B). Deletion of two adjacent nucleotides in the helical stem, UU6176-7, yielded a more severe defect on ribosome positioning, which correlated with the weaker translational activity associated with this mutation (Figure 2.4A, lanes 11-12 and 2.4B). Not surprisingly, deletion of two base-pairs within the helical stem, UU6176-7/AA6199-200, resulted in the complete abrogation of Toeprint A in accordance with the lack of translational activity (Figure 2.4A, lanes 13-14 and 2.4B). Thus, mutations within the helical stem resulted in a decrease in IRES activity through impairment of pseudoknot formation and ribosome positioning.  2.3.3 An optimal length of the variable loop region (VLR) is required to maintain robust IRES-mediated translation  The codon:anticodon mimicry domain of the IGR IRES is essential in establishing the translational reading frame to initiate translation from the ribosomal A site (Costantino et al., 2008; Sasaki and Nakashima, 2000; Wilson et al., 2000a). A variable loop region (VLR) interconnects the anticodon- and codon-like elements (Figure 2.5A), and was previously demonstrated to be susceptible to enzyme and chemical reagents that target single-stranded regions (Jan and Sarnow, 2002; Nishiyama et al., 2003). A comparison of the predicted secondary structural models for the IGR IRESs within the Dicistroviridae family reveals that the length of the VLR is not conserved and varies between six to ten nucleotides (Nakashima and Uchiumi, 2009). It has not been investigated whether the VLR length contributes to IRES activity. It is possible that the specific length of the VLR of each dicistrovirus IRES  54    Figure 2.4. Toeprinting analysis for IRESs bearing mutations in the helical stem. (A) Bicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in the absence [-] or presence [+] of purified salt-washed HeLa 80S (100 nM). Primer extension analysis was performed using PrEJ69 in the presence of α-[32P]-dATP. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography, as described in Section 2.2.4. The sequencing reactions for the wild-type IRES are shown on the left, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the right. (B) Summary of the translational activities and toeprint intensities for the mutant IRESs. Toeprint intensities were measured as a fraction of the radioactive counts for Toeprint A over the total radioactive counts within each lane, normalized to that of the wild-type IGR IRES.     55   Figure 2.5. Alterations in the length of the variable loop region (VLR) adversely affect IRES-mediated translation. (A) The schematic of the CrPV IGR IRES secondary structure is shown. The nucleotides constituting the VLR are highlighted in grey. The expected locations of a properly positioned ribosome on the IGR IRES (Toeprint A) and a translocated ribosome (Toeprint A+6 nt) are indicated. The first nucleotide of the codon occupying the ribosomal A site is indicated (+1). (B) Translational activities. Uncapped bicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [35S]-methionine, as described in Section 2.2.2. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments. (C) Summary of IRES translational activities and toeprint intensities in RRL for each construct. Insertion/ deletion mutations in the VLR are shown, with inserted and deleted nucleotides highlighted in grey and denoted with dash lines, respectively. Deviations from the wild-type length for each construct are indicated. Toeprint intensities were measured from Figure 2.6 (A).  56  may allow for optimal anticodon:codon-like interaction within PKI. To test this hypothesis, a series of insertion or deletion mutations were generated in this region by either introducing one to 12 arbitrarily selected nucleotides following U6211, the terminal nucleotide in the VLR of the CrPV IGR IRES, or deleting one or two nucleotides (Figure 2.5A and C). The deletions were made at two distinct locations within the loop to determine whether positional effects are associated with the deletions. Insertion of one, two or four nucleotides within the VLR moderately inhibited IRES activity (66-89% of wild-type IRES activity, Figure 2.5B and C). However, insertions in excess of four nucleotides exerted a more deleterious effect (9-31% of wild-type IRES activity, Figure 2.5B and C), with an increasingly more pronounced decrease in IRES translation observed with a greater increase in the length of the VLR. Deletion of one nucleotide within the VLR exhibited a positional effect, where the decrease in IRES activity associated with ΔU6203 was more drastic than was associated with ΔA6209 (30% versus 78%, Figure 2.5B and C). In both instances, however, a 2-nucleotide deletion exacerbated the inhibitory effects (Figure 2.5B and C). Thus, these observations indicate that the VLR can tolerate moderate increases but not decreases in length for optimal IRES-mediated translation.  To determine if repression of IRES-mediated translation for the VLR insertion/ deletion mutations is due to impaired ribosome positioning, toeprinting/ primer extension assay was performed in RRL. Reactions were performed in the presence of 15 µM edeine, which inhibits delivery of aminoacyl-tRNAs to the ribosomal A site (Carrasco et al., 1974; Szer and Kurylo-Borowska, 1970), or cycloheximide, which permits two elongation cycles during IRES-mediated translation (Jan and Sarnow, 2002; Pestova and Hellen, 2003; Wilson et al., 2000a). In accordance with previous findings, incubation of the wild-type CrPV IGR IRES with edeine generated a strong toeprint at nucleotides CA6226-7, indicative of a properly positioned ribosome on the IRES (Figure 2.6A, lane 2) (Jan and Sarnow, 2002; Wilson et al., 2000a). Toeprints were also observed at AA6161-2 (Toeprint B) and at A6182 (Figure 2.6A, lane 2), which indicate contacts established between the ribosome and the IRES that effectively impede passage of reverse transcriptase (Jan and Sarnow, 2002; Wilson et al., 2000a). Upon incubation with  57    Figure 2.6. Toeprinting analysis of VLR insertion/ deletion mutant IGR IRESs. Bicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in (A) a supplemented RRL without drug treatment [-], or treatment with either edeine [E] or cycloheximide [C] or (B) in the absence [-] or presence [+] of purified salt-washed HeLa 80S ribosomes (100 nM), as described in Section 2.2.4. The sequencing reactions for the wild-type IRES are shown on the left, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the right. Shown are representative gels from at least two independent experiments. 58  cycloheximide, a novel toeprint was observed at AC6232-3, 6 nucleotides downstream of Toeprint A, indicating that the ribosome has undergone two translocation cycles (Figure 2.6A, lane 3, Toeprint A+6 nt). Mutations that abolished the codon:anticodon-like base-pairing within PKI by introducing a CC6214-5GG mutation (ΔPKI) eliminated Toeprint A and the translocated toeprint, but not Toeprint B or at A6182 (Figure 2.6A, lanes 4-6). For single and double nucleotide deletions within the variable loop at A6209 and AU6209-10, treatment with edeine or cycloheximide generated an observable Toeprint A and a translocated toeprint (Figure 2.6A, lanes 25-30). In general, the decrease in intensities of Toeprint A and the translocated toeprint in the deletion mutants correlated with the decrease in IRES translational activities, albeit not as closely as that observed with the VLR insertion mutants (Figure 2.5C and 2.6A). For the VLR insertion mutations, Toeprint A observed upon edeine treatment decreased in intensity for insertions <4 nucleotides, but was essentially undetectable for insertions of or in excess of 4 nucleotides (Figure 2.6A, compare lanes 8 and 11 to 14, 17, 20, 23). Due to nucleotide insertions in the VLR, Toeprints B and at A6182 migrated slower in the sequencing gel as compared to that of the wild-type IGR IRES (Figure 2.6A). However and interestingly, cycloheximide treatment yielded a translocated toeprint that was observed for all insertion mutations even in the absence of Toeprint A, albeit with a trend of decreasing intensity that correlated with the observed translational activities (Figure 2.6A, lanes 9, 12, 15, 18, 21, 24). Thus, for the VLR insertion mutations, the intensity of Toeprint A+6 nt was more correlative with the observed translational efficiencies, as the intensity of Toeprint A approached background levels for constructs which still exhibited IRES activity (Figure 2.5C, Ins4, Ins7, Ins10, Ins12). To determine if a similar trend is observed with the positioning toeprints using only purified 80S ribosomes, toeprinting analysis was performed with salt-washed, purified HeLa 80S (Figure 2.6B). A comparable decrease in the intensities of Toeprint A with successive increases in the length of the VLR from 1 to 12 nucleotides was also observed (Figure 2.6B). Similar to that observed in Figure 2.6A, the decrease in Toeprint A intensities did not strictly correlate with the IRES translational efficiency (see discussion). In summary, these results indicate that an optimal length in the VLR is required for proper positioning of the ribosome on the IRES. 59  2.3.4 Sequence identity of the VLR impacts IRES-mediated translation  Although the length is not conserved, sequence alignment of at least six of nine members of the type I IGR IRESs indicates the presence of several conserved nucleotides in the VLR (Costantino et al., 2008) (Figure 2.7A). Specifically, nucleotides A6205, A6207, A6208 and U6211 of the CrPV IRES appear to be moderately conserved (Costantino et al., 2008). To determine if the sequence of the VLR is important for IRES-mediated translation, point mutations were introduced at the conserved bases, A6205, A6207, A6208, and U6211 of the CrPV IGR IRES and translational efficiency was assayed using the standard bicistronic reporter system (Figure 2.7B). Mutation of A6205 to each of the alternative Watson-Crick bases exhibited a moderate inhibition of IRES-mediated translation (Figure 2.7C, ~50% of wild-type). In contrast, mutation of A6207 exerted a negligible effect on IRES activity (Figure 2.7C). Interestingly, the effect on IRES translation by mutation of A6208 and U6211 was dependent on the nucleotide identity (Figure 2.7C). Mutating A6208 to G and U6211 to G moderately inhibited IRES translation, whereas U6211 to A slightly increased IRES activity by 30% (Figure 2.7C). Mutating A6208 and U6211 to other alternative nucleotides did not alter IRES translation (Figure 2.7C). These results demonstrate that A6205 is necessary for optimal IRES translation and that the nucleotide identity at A6208 and U6211, but not at A6207, affects IRES activity.  Because mutation at A6205 adversely affected IRES activity in a nucleotide-independent manner, a toeprinting/ primer extension assay was performed in a RRL to determine if mutation at this position conferred general defects in ribosome positioning and/ or translocation. As a control, ribosome positioning was monitored on mutant IRESs containing mutations at A6207, as mutations at this position did not affect IRES activity (Figure 2.7C). With edeine treatment, mutations at A6205 yielded a consistent decrease in the intensities of the positioning toeprint, with no significant decrease in the A6182 toeprint compared to the wild-type (Figure 2.8, lanes 5, 7, 9). Similarly, a decrease in the intensities of the translocated toeprints was also noted with cycloheximide treatment, consistent with the observed decrease in translational activities (Figure 2.8, lanes 6, 8, 10). For mutations at A6207, the intensities of the 60   Figure 2.7. The conserved A6205 is important for IRES-mediated translation. (A) Sequence alignment of the variable loop region (VLR) of members of the Type I IGR IRESs. Nucleotides involved in base-pairing of the helical stem are bolded and those comprising pseudoknot I are highlighted in grey. The nucleotides constituting the VLR are denoted, with conserved positions highlighted in red. The assignments are made based on the secondary structural predictions of the IGR IRESs (Nakashima and Uchiumi, 2009). The translational start site is indicated. (B) A schematic of the PKI domain is shown with the nucleotides constituting the VLR highlighted in grey. Four conserved nucleotides within the VLR, A6205, A6207, A6208 and U6211 are denoted by boxes and were mutated to each of the alternate Watson-Crick bases. (C) Translational activities of VLR point mutants. Uncapped bicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [35S]-methionine, as described in Section 2.2.2. Shown are the average values ± S.D. for the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES from at least three independent experiments.    61    Figure 2.8. Toeprinting/ primer extension assay of VLR point mutants. Bicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in RRL under treatment with edeine [E] or cycloheximide [C], as described in Section 2.2.4. Primer extension analysis was performed using PrEJ69 in the presence of α- [32P]-dATP. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The sequencing reactions for the wild-type IRES are shown on the left, with the respective nucleotide numbers as indicated. The locations of the major toeprints are as indicated on the right. Shown is a representative gel from at least two independent experiments.   62  positioning and translocated toeprints observed with edeine and cycloheximide treatment were similar to wild-type and corresponded to the observed translational activities (Figure 2.8, lanes 11-16). Thus, these results suggest that A6205 but not A6207 has a role in ribosome positioning. It remains to be investigated whether mutations at A6205 alter the structure of the PKI domain, which in effect may impair ribosome positioning on the IRES.  2.3.5 IRES-mediated translation initiation is permissible at an adjacent and overlapping alternate start site  In canonical translation initiation, recognition of the translational start site occurs through optimal codon:anticodon interaction involving initiator Met-tRNAMet·eIF2·GTP ternary complex . During factor-independent IGR IRES-mediated translation, the PKI domain of the IRES plays an analogous role by precisely mimicking an authentic codon:anticodon pairing in the ribosomal P site and positioning the initiation codon in the A site to receive the first incoming aminoacyl-tRNA (Costantino et al., 2008; Wilson et al., 2000a). Previous biochemical studies have suggested that interactions within PKI are dynamic (Jan and Sarnow, 2002; Nishiyama et al., 2003). Furthermore, recent data suggest that a subset of IGR IRESs can initiate translation in an alternate reading frame through an IRES-dependent mechanism (Ren et al., 2012). These observations suggest that proper reading frame selection for IRES-mediated translation initiation may be intrinsic within the tRNA-like domain. Thus, translation initiation at an alternate start site may be permissible provided that critical base-pairing interaction within PKI are maintained.   To test this, UACCU6212-6, which constitutes the codon-like element of PKI, was duplicated and inserted either two nucleotides downstream of the authentic codon-like element, or immediately adjacent to and overlapping by one nucleotide (Figure 2.9, 2ndsite and 2ndsite Adj, respectively). Because the inserted sequence exhibited base complementarity to the anticodon-like element, it provided an alternate site for PKI base-pairing. The inserted sequence was introduced into an alternate reading frame; 63  consequently, two distinct reporter constructs were generated to differentiate between selection of the authentic (highlighted blue box) or the inserted translational start sites (highlighted in yellow box) (Figure 2.9). Where necessary, the appropriate mutations were made near the reporter start codon such that initiation from the authentic or inserted start sites could be monitored by FLuc expression. Within these bicistronic reporter constructs, IRES-mediated FLuc translational activity was normalized to the upstream RLuc, which served as the normalization control between experiments.  For the construct where the codon-like element was inserted two nucleotides downstream of the authentic site, negligible reporter expression from the inserted site was observed (Figure 2.10, 2ndsite, yellow box), in contrast to robust IRES translation observed from the authentic site (Figure 2.10, 2ndsite, blue box). To determine if the authentic and inserted sites competed for base-pairing with the anticodon region of PKI, a CC6214-15GG substitution mutation was introduced (Figure 2.9, 2ndsite CC6214-15GG), which effectively disrupted base-pairing at the authentic site to potentially promote base-pairing exclusively at the inserted sequence. Interestingly, the CC6214-15GG mutation resulted in negligible FLuc expression (Figure 2.10, 2ndsite CC6214-15GG, yellow box), suggesting that the authentic translational start site may be preferentially selected in IRES-mediated translation. When the second codon-like element was introduced adjacent to the authentic site, translation from the inserted site was observed (9-14% of the wild-type IGR IRES activity) (Figure 2.10, 2ndsite Adj, yellow box). Although within this construct, the authentic site supported only ~51% of the wild-type IGR IRES activity (Figure 2.10, 2ndsite Adj, blue box), the extent of IRES translation was higher compared to the inserted site, further suggesting an inherent preference for the authentic translational start site even when an alternate site is accessible (Figure 2.10, 2ndsite Adj, compare yellow and blue boxes). Disrupting the authentic site by introducing the CC6214-15GG mutation did not significantly affect translation from the adjacent inserted site (Figure 2.10, 2ndsite Adj, CC6214-15GG). Taken together, these findings indicate that IRES-mediated translation initiation is permissible at an alternate start site, but only when the inserted site is located adjacent to the authentic site.  64    Figure 2.9. IGR IRES-mediated translation initiation at an alternate translational start site. Top. Toeprinting analysis for the respective constructs. Bicistronic reporter RNAs containing the wild-type or mutant IGR IRES were incubated in RRL without drug treatment [-], or under treatment with edeine [E] or cycloheximide [C], as described in Section 2.2.4. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The sequencing reactions for the constructs are shown, with the nucleotides corresponding to the authentic and inserted sites highlighted in blue and yellow, respectively. The locations of the major toeprints are as indicated to the right of the gel in the same color. Bottom. Schematic of the construct of the wild-type IGR IRES PKI domain and constructs which contain an alternate translational start site 2 nucleotides downstream [2ndsite], or adjacent to and overlapping with the authentic start site [2ndsite Adj]. The authentic and inserted translation start sites are highlighted in blue and yellow, respectively. The expected locations of a properly positioned ribosome (Toeprint A) and a translocated ribosome (Toeprint A+6 nt) from the authentic and inserted sites are denoted by arrows of the corresponding colors for each construct.  65   Figure 2.10. IRES translational activities of reporter constructs containing alternate translational start sites. Uncapped bicistronic reporter RNAs containing the wild-type or mutant IGR IRES in the intercistronic space were incubated in RRL in the presence of [35S]-methionine, as described in Section 2.2.2. Shown are the ratios of FLuc to RLuc expression, normalized to the wild-type IGR IRES. Translational activities monitored in the reading frame of the authentic and inserted start sites are highlighted in blue and yellow, respectively. Shown are average values from at least three independent experiments ± S.D.   66  To confirm that ribosomes are in fact positioning at both sites of initiation, toeprinting analysis was performed in RRL (Figure 2.9). For the 2ndsite construct, treatment with edeine generated a single observable toeprint at nucleotides AU6226-7, indicating a properly positioned ribosome at the authentic translational start site (Figure 2.9, lane 8). Treatment with cycloheximide generated a translocated toeprint at nucleotides AG6232-3, six nucleotides downstream, which was consistent with a ribosome positioned after two cycles of translocation (Figure 2.9, lane 9). Provided that ribosomes were positioned at the inserted site of the 2ndsite construct, toeprints should be observed at GA6233-4 and AU6239-40 under edeine and cycloheximide treatment, respectively (Figure 2.9, 2ndsite, bottom). However, no detectable toeprints were observed with edeine or cycloheximide treatment at either of these sites even when the authentic site was disrupted (Figure 2.9, lanes 10-12), consistent with the observation that IRES-mediated translation initiated exclusively from the authentic codon-like element. For the 2ndsite Adj construct, a single discrete toeprint, A, corresponding to a properly positioned ribosome at the authentic translational start site was observed at nucleotides AU6226-7, which is inconsistent with the observation that both sites supported translation (Figure 2.9C, lane 14). Only upon treatment with cycloheximide were two distinct toeprints discernible: a higher intensity toeprint at AG6232-3 and a lower intensity toeprint at AC6236-7 which corresponded to translocation from the authentic and inserted sites, respectively (Figure 2.9, lane 15). Similarly, the 2ndsite Adj CC6214-5GG construct, which supported translation initiation exclusively from the inserted site, generated an observable toeprint at AC6236-7 only with cycloheximide, but not edeine treatment (Figure 2.9, lanes 16-18). In summary, these results indicate that although translation initiation mediated by the CrPV IRES is permissible at an alternate start site, optimal translation initiation occurs at the authentic start site.  2.4 Discussion High-resolution structural investigations of the CrPV IGR IRES PKI domain have yielded significant insights into the remarkable ability of structured RNAs to manipulate the translational machinery (Costantino et al., 2008; Zhu et al., 2011). To complement previous structural and biochemical 67  studies, we have used novel mutations to probe the tRNA mimicry domain to identify specific elements that confer IRES activity and the ability to recruit the ribosome and select the translational reading frame. Deviations in the length of the PKI helical stem, VLR, or the anticodon:codon base-pairing resulted in reduced IRES translation, suggesting that the tRNA-mimicry domain of the wild-type IRES has been optimized for IRES translation. Furthermore, we have identified specific nucleotides within the VLR that contribute to IRES activity. These studies have revealed the structural constraints and parameters of the anticodon mimicry domain for IRES-mediated translation and provide a molecular framework for understanding how the IGR IRES hijacks and engages the ribosome into an elongation-competent mode. The disordered nature of the VLR in the crystal structure of the IRES PKI domain bound to the ribosome is a conserved feature for both the CrPV and the related Plautia stali intestine virus (Zhu et al., 2011). Here, we demonstrate that the wild-type CrPV IGR IRES has preserved an ideal length of the VLR for translation (Figures 2.5 and 2.10). The reduced translational activities of the VLR insertion mutants and mutants bearing alternate start sites suggest that there are constraints within the VLR and PKI domain that limit the range for base-pairing for pseudoknot formation and reading frame selection by the IGR IRES (Figure 2.5 and 2.10). While a robust positioning toeprint was noted for the wild-type IRES, translationally active mutants bearing VLR insertions generated observable toeprints only upon translation elongation (Figure 2.6). A similar phenomenon was also observed when IRES translation initiated from an adjacent start site, which effectively increases the length of the VLR (Figure 2.9). These results suggest that the additional sequences within the VLR – either by nucleotide insertion or selection of the downstream alternate start site – may impede optimal docking of the IRES into the ribosomal A site, possibly due to steric hindrance, although this remains to be tested. Alternatively but not exclusively, increasing the length of the VLR may promote more transient interactions between the IRES and the ribosome such that the reverse transcription reaction cannot sufficiently capture the positioning toeprint during primer extension. In both types of mutations, the translocated toeprint was observed with cycloheximide treatment, suggesting that delivery of the Ala-tRNAAla to the ribosomal A site and 68  subsequent translocation steps further stabilize the IRES-80S complex (Figure 2.6 and 2.9). Although further experimentation will be required to differentiate between these two models, the discrepancy between the positioning toeprint to the IRES translational activities observed here underscores the need to revisit the previous use of the positioning toeprint as a diagnostic for translational activity (Jang and Jan, 2010; Jang et al., 2009).  We have recently demonstrated that a subset of IGR IRESs can initiate translation in the +1 reading frame through a non-canonical base-pair adjacent to PKI (Ren et al., 2012). Here, our data hints at the propensity of the CrPV IGR IRES to initiate translation at a different start site and potentially in an alternate reading frame, as an overlapping and adjacent codon-like element supported IRES-mediated translation (Figure 2.10). However, more robust activity was consistently observed from the authentic translational start site in both the translation and toeprinting assays, indicating an optimal context for IRES-mediated translation (Figure 2.9 and 2.10). Interestingly, the lack of increase in IRES translation from the alternate site when the authentic site was compromised by base substitution suggests little competition between initiation from the two sites (Figure 2.10). The dynamic nature of the tRNA-mimicry domain, supported by structural and enzymatic probing data, may enable the IRES to sample for the appropriate translational reading frame through base-pairing between the anticodon- and codon-like elements (Costantino et al., 2008; Jan and Sarnow, 2002; Nishiyama et al., 2003). It will be of interest to further examine the implications of the dynamic nature of the PKI domain on IRES-mediated translation and to determine whether the differences in the VLR lengths of other dicistroviruses are also ideal for IRES translation.  Our results indicate that the nucleotide identity within the VLR has a role in IRES activity. Translation initiation at the adjacent or downstream alternate translational start sites effectively increases the length of the VLR by 4 or 7 nucleotides, respectively (Figure 2.10). While the data are complementary, the differences in activities of the 2ndsite Adj and 2ndsite mutants with VLR mutants harboring 4- and 7-nucleotide insertions imply that the sequence of the VLR is a critical determinant of IRES activity. In 69  support of this, mutating the conserved A6205, and A6208 and U6211 to specific nucleotides resulted in a decrease in IRES activity (Figure 2.7). It remains to be investigated whether A6205 or other nucleotides within the VLR affect the conformation of PKI or may interact, possibly transiently, with the ribosome, IRES and/or the incoming tRNA to mediate IRES-dependent translation. Structural comparison between the anticodon mimic of the IRES and an authentic tRNA anticodon:codon pair reveals remarkable similarities, although some deviations occur in the vicinity of the PKI base-pairs (Costantino et al., 2008). Most notably, the three bases constituting the anticodon in the initiator tRNA have been replaced by five analogous base-pairing interactions within PKI, and some structural rearrangements are introduced which are not observed in the authentic tRNA-mRNA interaction (Costantino et al., 2008). Our results demonstrate that an authentic tRNA anticodon stem-loop can impart limited translational activity (Figure 2.2). Additionally, abrogation of the additional base-pairs within PKI of the CrPV IRES – by mutation of UA6190-1 or UA6212-3 to their Watson-Crick complements such that the base-pairing more closely resembles a true codon:anticodon pairing – suggests that three base-pairing interactions can confer IRES translation, albeit weakly (Figure 2.2). Additional base-pairs within PKI likely evolved to further modulate IRES translational efficiency. For instance, deletion of A6191 results in an enhancement in IRES translation, suggesting that IRES activity is not maximal but may be moderated for virus infection (Costantino et al., 2008). Our results also demonstrated the modular nature of the IGR IRES. This observation is not unprecedented, as derived IRESs comprised of the ribosome-binding and tRNA mimicry domains from two distinct IGR IRES classes can mediate IRES translation (Hertz and Thompson, 2011; Jang and Jan, 2010). The results herein present an enticing possibility that the IRES may have originated from tRNA-like elements or evolved from the anticodon stem-loop of an authentic tRNA, and subsequently acquired mutations that enable it to function optimally for viral IRES-mediated translation. Following the completion of this work, another group has independently pursued the function of the VLR using biochemical and biophysical approaches (Ruehle et al., 2015). More drastic mutations 70  were introduced into the VLR, including further shortening of the loop by three nucleotides to restrict flexibility, and reducing the high adenosine content by substitution with guanosine. These mutations all rendered the IRES translationally inactive. While our studies featured more subtle mutations, the intentional focus on mutants that exhibited more pronounced translational defects, complemented with quantitative kinetic assays, led the authors of this recent study to suggest an additional intrinsic role of VLR in non-canonical translocation. Specifically, toeprinting and anisotropy data suggest that VLR shortening impairs the translocation of PKI from the A site to the P site and thus interferes with aminoacyl-tRNA binding (Ruehle et al., 2015). By contrast, altering the base composition of the VLR such that it is more guanosine-rich inhibits the second pseudo-translocation step that involves displacement of the first aminoacyl-tRNA to the P site (Ruehle et al., 2015). While these observations are provoking and may suggest multiple roles of the VLR in promoting pseudo-translocation, there is the obvious caveat - as with all mutational analysis studies - that such mutations may give rise to artefacts. It will be interesting to assess if the same mutations in the VLR will impair viral infection. Recent cryo-EM structures of IRES/ribosome complexes have also provided some structural insights into the localization of the VLR (Fernandez et al., 2014; Koh et al., 2014; Murray et al., 2016). The poor resolution of the VLR observed for ribosome-bound CrPV and TSV IGR IRESs in a pre-translocation state is consistent with the conformational flexibility of this region (Fernandez et al., 2014; Koh et al., 2014). However, partial density of the VLR can be observed for the 40S/CrPV IRES binary complex, which may suggest a potential role of the VLR in 40S recruitment and/or positioning of the anticodon:codon mimic (Murray et al., 2016). Similarly, the VLR adopts an ordered conformational state following translocation by binding in the 40S E site, with the backbone proximal to the codon-like element interacting with uS7 (Abeyrathne et al., 2016; Muhs et al., 2015). Ordering of the VLR in the post-translocation state hints at the possibility that conformational rearrangement may help stabilize the PKI upon translocation in the P site. Our findings, along with recent biophysical and structural data, suggest that the VLR has multiple intrinsic roles in IRES-mediated translation initiation. 71  Viruses have evolved unique mechanisms for translating their genomes as a strategy to hijack the ribosome during infection when cap-dependent translation is compromised. Both HCV and CrPV IGR IRESs have acquired a pseudoknot to directly manipulate the ribosome and position it properly at the initiation codon; however, each IRES utilizes the pseudoknot distinctly. The HCV pseudoknot acts as a connector region to link the ribosome binding domain and the initiation codon-containing domain and to orient the HCV initiation codon in the mRNA binding cleft of the 40S subunit to presumably mediate binding of the ternary complex eIF2-GTP-Met-tRNAi (Berry et al., 2011). In contrast, the IGR IRES pseudoknot does not require any factors. The PKI domain connects the anticodon stem-loop-like element with the initiation codon in cis, and translocates into the ribosomal P site to engage the ribosome to initiate translation from the A site. For both IRESs, the molecular framework of the pseudoknot domains has been optimized for efficient IRES translation (Berry et al., 2011), this work). Given the use of pseudoknot elements in such divergent IRES mechanisms, it will be intriguing to consider if pseudoknot structures represent a more widespread strategy utilized by other IRESs for ribosome positioning and reading frame selection. Finally, it is interesting to note that some plant RNA viruses also utilize a tRNA-like element within their 3'UTR to mediate viral translation and replication, suggesting that tRNA mimicry may be a more general strategy for viral translation (Colussi et al., 2014; Dreher and Miller, 2006; Matsuda and Dreher, 2004). Identifying additional interacting partners of the VLR, and understanding coordinated changes in its interaction with specific ribosomal components may yield further insight into its role in IRES-mediated translation.    72  CHAPTER 3: GLOBAL SHAPE MIMICRY OF tRNA OF THE ISRAELI ACUTE PARALYSIS VIRUS INTERGENIC REGION INTERNAL RIBOSOME ENTRY SITE MEDIATES TRANSLATIONAL READING FRAME SELECTION 3.1 Introduction  Fidelity of protein synthesis and the transmission of genetic information from mRNA into a nascent protein rely on the accurate selection and maintenance of the translational reading frame. In canonical eukaryotic translation, after recruitment and scanning of ribosomes on an mRNA, the translational reading frame is initially established by Met-tRNAi anticodon:codon pairing in the ribosomal P site following the scanning process. Once the reading frame has been selected during initiation, it is normally maintained as the default condition with high fidelity. Nonetheless, programmed recoding has been identified in cellular and viral mRNAs. Such events rely on cis-acting signals and permit an increase in coding capacity by allowing translation to use alternative reading frames (Dinman, 2012; Firth and Brierley, 2012).  We have recently demonstrated that a subset of viruses within the Dicistroviridae family harbors an intergenic region internal ribosome entry site (IGR IRES) that can direct translation in alternative reading frames (Ren et al., 2012), thus providing an excellent model for studying RNA/ribosome interactions that influence reading frame selection. The overlapping +1 open reading frame (ORFx) was identified in several Type II dicistrovirus IRESs including the honeybee viruses (Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and Acute bee paralysis virus (ABPV)) and a fire ant virus (Solenopsis invicta virus-1 (SINV-1)) (Firth et al., 2009; Sabath et al., 2009). Through extensive mutagenesis, we demonstrated that translation of ORFx initiates via a U:G wobble adjacent to the IRES translational start site (Ren et al., 2012); however, certain mutations within the IRES PKI domain can relieve the dependence on the wobble base-pair and effectively uncouple the translational selectivity by the IRES to initiate either in the 0 or +1 frames (Ren et al., 2014). These findings suggest that the IGR 73  IRES adopts varied conformations that facilitate translational reading frame selection and that some PKI mutations may promote a predominate conformation that directs exclusive 0 or +1 frame translation. Consistent with this, RNA structural probing analyses have identified subtle conformational rearrangements (Ren et al., 2014). While the functional contribution of ORFx to virus infection remains to be fully elucidated, the discovery of an IRES-dependent mechanism that effectively increases the coding capacity of compact viral genomes is unprecedented.  Type II IGR IRESs can be differentiated from Type I IRESs based on the presence of characteristic structural elements (larger loop L1.1 and additional stem-loop (SLIII)), although the function of these extra features is not well understood (Figure 3.1). Because Type I and II IGR IRESs adopt overall comparable secondary structures, they generally have been considered functionally and mechanistically similar (Cevallos and Sarnow, 2005; Hatakeyama et al., 2004; Pfingsten et al., 2007). Indeed, using chimeric IRESs, the ribosome binding and PKI domains of Type I and II IRESs are functionally interchangeable (Hertz and Thompson, 2011; Jang and Jan, 2010). Recent cryo-EM structures of the Taura syndrome virus (TSV) Type II IRES bound to the ribosome confirmed that similar interactions with the ribosome are shared between Type I and II IRESs (Koh et al., 2014). Interestingly, the TSV SLIII, which is stacked coaxially on PKI, extends towards the large ribosomal subunit of the A site (Au et al., 2015; Koh et al., 2014). Disruption of SLIII base-pairing inhibits TSV IRES translation, which can be effectively restored by compensatory mutations (Hatakeyama et al., 2004). Similarly, deletion of SLIII abrogates IRES activity and proper ribosome positioning on the IRES, but does not affect ribosome binding (Jang and Jan, 2010; Pfingsten et al., 2007). While such findings indicate that SLIII is indispensable, its exact role in IRES-mediated translation has not been unambiguously defined. In this study, we provide insights into the function of SLIII of the Type II IAPV IGR IRES using biochemical and structural approaches. Structural studies using a nuclear magnetic resonance/ small angle X-ray scattering (NMR/SAXS) hybrid approach reveal that the PKI domain resembles a complete tRNA. Moreover, mutagenesis studies and structural probing experiments demonstrate that certain mutations  74     Figure 3.1. Secondary structure of the IAPV IGR IRES. Pseudoknots (PKs) I, II and III, stem loops (SL) III, IV and V, loop L1.1 and the variable loop region (VLR) are indicated. The PKI domain is comprised of a three-way junction involving helices P3.1 (green), P3.2 (purple) and P3.3 (blue). The IAPV IRES can mediate translation of ORFx in the +1 reading frame which overlaps the viral structural protein coding region.  IRES-mediated translation in the 0 and +1 frames starts from the GGC glycine and GCG alanine codons, respectively. Translation of the +1 frame ORFx is directed by a U6562/G6618 base-pair adjacent to PKI (red nucleotides).   75  within the SLIII element result in an uncoupling of 0 and +1 frame translation that correlate with different IRES conformations. These studies provide insight into how the PKI domain of the IAPV IRES mimics a complete tRNA to gain access to the tRNA-binding domains of the ribosome to mediate reading frame selection during IRES translation initiation.   3.2 Materials and methods 3.2.1 Plasmids and constructs The monocistronic and bicistronic luciferase plasmids containing the IAPV IGR IRES have been described previously (Jan and Sarnow, 2002). Mutant IRESs were generated by PCR-based site-directed mutagenesis and verified by sequencing. Monocistronic luciferase plasmids used for selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) analysis and bicistronic luciferase plasmids used for translation and toeprinting/primer extension assays were digested with NarI and XbaI, respectively (see Appendix A).  3.2.2 In vitro translation assays Bicistronic luciferase plasmids were linearized and incubated in an Sf21 transcription/translation-coupled extract (Promega) containing an additional 20 mM potassium acetate and 0.5 mM magnesium chloride (final concentration). Reactions were performed in the presence of 3 µCi [35S]-methionine/cysteine (Perkin Elmer) at 30°C for 2 h and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and phosphorimaging (Typhoon, Amersham).  3.2.3 Purification of 40S and 60S ribosomal subunits Purification of ribosomal subunits was performed as described in Section 2.2.3 Ribosomal subunits were purified from HeLa cell pellets (National Cell Culture Center). HeLa cells were lysed in lysis buffer (15 mM Tris-HCl (pH7.5), 300 mM NaCl, 1% (v/v) Triton X-100, 6 mM MgCl2, 1 mg/ml heparin) and the supernatant was centrifuged briefly to remove cellular debris. The supernatant was transferred to a 30% 76  (w/w) sucrose cushion containing 500 mM KCl and centrifuged at 100,000 g to pellet ribosomes, which were subsequently resuspended in buffer B (20 mM Tris-HCl (pH 7.5), 6 mM magnesium acetate, 150 mM KCl, 6.8% (w/w) sucrose, 2 mM DTT). To release ribosomes from translating mRNAs, puromycin was added and the final concentration of KCl was increased to 500 mM to dissociate the ribosomal subunits. The dissociated ribosomes were separated on a 10-30% (w/w) sucrose gradient and the absorbance was monitored at 260 nm to detect the free 40S and 60S subunits. The corresponding fractions were collected, pooled, and concentrated in buffer C (20 mM Tris-HCl (pH 7.5), 0.2 mM EDTA, 10 mM KCl, 1 mM MgCl2, 6.8% (w/w) sucrose) using Amicon Ultra spin concentrations (Millipore). The concentrations of the free ribosomal subunits were determined by spectrophotometry using the conversions 1 A260 nm = 50 nM and 1 A260 nm = 25 nM for the 40S and 60S subunits, respectively. 3.2.4 Toeprinting/primer extension assay Toeprinting analysis was performed as previously described (Wilson et al., 2000a). 150 ng of bicistronic wild-type or mutant IRES RNAs were annealed to primer PrEJ761 (5'- CATGGGGGTATCGATCCTATTTGGAG-3') in 40 mM Tris-HCl (pH 7.5) and 0.2 mM EDTA by heating to 65°C, followed by slow cooling to 37°C. The annealed RNAs were incubated with 100 nM and 150 nM final concentration of purified 40S and 60S subunits, respectively. Ribosome positioning was analyzed by primer extension/reverse transcription using 10 units of AMV reverse transcriptase (Promega) in the presence of 125 µM of each of deoxythymidine  triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate, 25 µM deoxyadenosine triphosphate, 0.5 µL of α- [32P] dATP (3.33 µM, 3000 Ci/mmol), 8 mM MgOAc, 10 units of Ribolock, 1X buffer E (20 mM Tris-HCl,(pH 7.5), 100 mM KCl, 2.5 mM MgOAc, 0.25 mM spermidine, 2 mM DTT) in the final reaction volume. Reverse transcription was performed at 37°C for 1 h, after which the reaction was quenched by extraction with phenol/chloroform (twice), chloroform (once) and ethanol precipitated. The resultant complementary DNA fragments were analyzed by 6% (w/v) denaturing gel electrophoresis. The gels were subsequently dried and subjected to phosphorimager analysis.  77  3.2.5 Reconstitution of IRES-mediated translation To reconstitute translation in vitro, IRES-ribosome complexes were assembled as described above, in the presence of 1 mM ATP, 0.4 mM GTP and 0.5 mg/mL cycloheximide. Following incubation, purified yeast elongation factor 1A (30 ng/µL), elongation factor 2 (50 ng/µL), and bulk bovine aminoacyl-tRNAs were added to promote translocation. Ribosome positioning was determined by reverse transcription, as described above. For reconstitution experiments using eukaryotic release factor 1 (eRF1), purified, salt-washed human ribosomes were assembled on IRES RNAs in the presence of 0.5 mM GTP, followed by the addition of yeast elongation factor 2 (50 ng/µL) and eRF1 (50 ng/µL). Reverse transcription is performed as described above.  3.2.6 RNA structural probing 10 pmol of monocistronic RNA bearing the wild-type or mutant IRESs was briefly heated to 95°C, followed by slow cooling to 30°C for 20 min in Buffer E (final concentration of 20 mM Tris, pH 7.5, 100 mM KCl, pH 7.0, 2.5 mM MgOAc, 0.25 mM spermidine, 2 mM DTT) to induce folding. The RNA was modified by addition of 3 mM (final concentration) N-methylisatoic anhydride (NMIA) solubilized in DMSO, or in parallel with treatment with neat DMSO (as the -NMIA control). The reaction was performed at 30°C for 90 min and the modified RNA was recovered by ethanol precipitation, as described previously (Wilkinson et al., 2006). For dimethyl sulfate (DMS) modification,  the RNA was treated with 1:400 dilution (in 100% ethanol) of DMS for 10 min at 30°C, in the presence of bulk yeast tRNAs. Following incubation, the reaction was terminated by the addition of quench buffer (30% (v/v) β-mercaptoethanol, 300 mM NaOAc, pH 5.0) (Tijerina et al., 2007) and the modified RNAs were recovered by ethanol precipitation. For both NMIA- and DMS-modified RNAs, primer extension was performed using equimolar 5'-end labelled primer PrHA40:  5'-AGTTCCGTATTGTGTACGTTGGGAG-3' and reactions were subsequently analyzed by 8% (w/v) denaturing polyacrylamide gel electrophoresis. Individual band intensities were quantitated by Semi-Automated Footprint Analysis (SAFA) and 78  normalized as described previously (Das et al., 2005; McGinnis et al., 2009). Band intensity variations for each nucleotide were normalized by dividing all intensities by the average value of the next 10% of reactive nucleotides, after excluding the top 2% of the most reactive nucleotides.   3.3 Results 3.3.1 The integrity of SLIII is important for IRES-mediated translation  Previous reports indicated that the PKI domain of the Type II IGR is important for IRES translation (Hatakeyama et al., 2004; Jang and Jan, 2010; Pfingsten et al., 2007); however, the function of SLIII has not been examined in detail. We first addressed whether distinct regions of SLIII, specifically the apical loop and helix P3.3, are important in IRES-mediated selection of the translational reading frame using a bicistronic reporter assay, as described previously (Ren et al., 2012) (Figure 3.2A). IRES-mediated +1 frame translation is approximately 20% of 0 frame translation in an in vitro Sf21 transcription-translation system (Ren et al., 2012). For simplicity, we have normalized all IRES translation activities to the wild-type 0 and +1 frame translational activities set as 100%. Mutation of the nucleotides within the apical loop yielded negligible effects in IRES-mediated translation initiation in the 0 and +1 reading frames, indicating that the nucleotide identities in this loop are not essential for IRES activity (Figure 3.2B, orange box).   To determine if the integrity of helical stem P3.3 is important for IRES activity, base-pair deletions were systematically tested. Shortening the helical stem by deletion of one base-pair at various positions (Figure 3.3A, constructs (i)-(iv)), moderately inhibited 0 frame translation by 23-55% but stimulated +1 frame translation by 13-20% compared to the wild-type IRES (Figure 3.3B, Δ1 bp). Deletion of ΔA6582/ΔU6593 resulted in a stronger defect in 0 frame translation (55% decrease), suggesting that the identity or position of this base-pair may be important for IRES translation (Figure 3.3, construct (iv)). Further shortening of helix P3.3 by deletion of two base-pairs (constructs (v)-(vii)) or three base-pairs (constructs (viii)-(ix)) exacerbated IRES-mediated 0 frame translation, and IRES activity  79    Figure 3.2. Translational activities of IAPV IRES PKI mutants. (A) Bicistronic reporter construct. The bicistronic reporter contains an upstream Renilla luciferase reporter (Ren Luc) and downstream firefly luciferase reporter (FLuc), which are expressed by scanning-dependent and IRES-dependent translation, respectively. FLuc is fused in the +1 reading frame, downstream of the ORFx coding sequence. 0 frame translation results in a truncated protein (sORF2). (B) Summary of translational activities of wild-type and mutant IRESs. Translational activities, determined as described in Section 3.2.2, are normalized to the wild-type IRES, which is set to 100% for both the 0 and +1 frames. For the wild-type IRES, +1 frame translation is approximately 20% of 0 frame translation in vitro. Shown are the average values from at least three independent experiments ± 1 s.d. Asterisk denotes data that have been previously published (Ren et al., 2014). Translation assays for the SLIII apical loop mutants (boxed in orange) were performed by Seonghoon Lee.    80    Figure 3.3. The integrity of stem-loop (SL) III is important for 0 frame translation. (A) Schematics of IRES mutants harboring systematic one base-pair (i-iv), two base-pairs (v-vii) or three base-pairs deletions (viii-ix) at various positions along helix P3.3 of SLIII. (B) Relative translational activities of helix P3.3 deletion mutants, expressed as a ratio of 0 frame translation to Ren Luc expression (left), or a ratio of +1 frame translation to Ren Luc expression (right). Translational activities for some deletion mutants were measured by Seonghoon Lee.    81  was essentially abolished for construct (ix) (Figure 3.3). Interestingly, while a defect in 0 frame translation was observed, +1 frame translation was increased by 42-202% with these mutations, suggesting that the integrity of helix P3.3 is important for 0 frame, but not +1 frame translation. These mutations and especially (ix), resulted in the uncoupling of 0 and +1 frame translation, as observed previously with other point mutations within the PKI domain (Ren et al., 2014).   Disruption of helix P3.3 base-pairing by base substitution yielded a significant defect in 0 frame translation, with varying effects on +1 frame translation (Figure 3.4, (i)-(ii), (v)-(vii) and (ix)). Restoration of base-pairing interactions through compensatory mutation was sufficient to rescue 0 frame translation to levels comparable to or exceeding that of wild-type within some contexts, suggesting that optimal 0 frame translation is dependent on both SLIII base-pairing and nucleotide sequence (Figure 3.4, constructs (iii), (iv) and (viii)). Surprisingly, disruption of a single base-pair interaction reduced 0 frame translation to ~25% of wild-type (Figure 3.4, construct (x)). In summary, these results indicate that the integrity of P3.3 is essential for 0 frame translation.  3.3.2 Ribosome positioning is unaffected in SLIII mutants  To determine if translational defects observed with SLIII mutants are due to an impairment in ribosome positioning, toeprinting assays were performed using a representative subset of the helix P3.3 mutants. In the toeprinting assay, the primer extension reaction arrests upon encountering the 3' edge of the ribosome, generating cDNA fragments (or 'toeprints') that can be resolved on a sequencing gel. Purified, salt-washed HeLa ribosomes assembled on the wild-type IAPV IRES resulted in a discrete toeprint at A6628 (+14 position) as observed previously (Ren et al., 2014), where C6615 is designated as +1 (Figure 3.5A, lanes 1-2). This toeprint represents a ribosome positioned at the IRES translational start site with the PKI domain in the ribosomal A site (Fernandez et al., 2014; Koh et al., 2014). The +14 A6628 toeprint was not observed for an IRES mutant deficient in ribosome positioning (ΔPKI) (Figure 3.5A, lanes 3-4), demonstrating that the toeprinting assay provides a specific assessment of proper  82    Figure 3.4. Translational activities of helix P3.3 mutants. Mutations were introduced at two positions along helix P3.3 of SLIII, as indicated on the PKI secondary structure. Specific mutations at each position are denoted by boxes in the corresponding color, with mutated bases shown in red. Translational activities were determined as described in Section 3.2.2. Relative translational activities of the helix P3.3 mutants are expressed as a ratio of 0 frame translation (sORF2) to Ren Luc expression, or as a ratio of +1 frame translation (ORFx) to Ren Luc expression, normalized to the wild-type IRES set as 100% for both reading frames.   83    Figure 3.5. Toeprinting/primer extension analysis of helix P3.3 mutants. Toeprinting analysis of IAPV IRES/ribosome complexes for (A) helix P3.3 deletion mutations as depicted in Figure 3.3 or (B) A6576 mutants. Bicistronic RNAs harboring the wild-type or mutant IRESs were incubated alone (-) or with salt-washed, HeLa ribosomes (+) and analyzed by primer extension, as described in Section 3.2.4. The sequencing reactions of the wild-type IRES are shown on the left, with the position of the +1 nucleotide indicated for reference. The position of the observed toeprint is as denoted. Quantitation of the toeprints is shown for helix P3.3 mutants (C) and A6576 mutants (D). 84  ribosome positioning on the IRES (Ren et al., 2014). With the exception of the three base-pair deletion mutant (ix), all SLIII base-pair deletion mutants yielded toeprints that were comparable in intensity to the wild-type IRES, suggesting that the translational defects observed with these mutants are likely not due to impaired ribosome positioning but a downstream step (Figure 3.5A, lanes 5-16 and 3.5C). For the three base-pair deletion mutant (ix), the approximate 60% loss in toeprint intensity suggests that impairment may be occurring at the step of ribosome binding, positioning and/or at a downstream step, which all together manifests as the complete loss of translational activity within the 0 frame (Figure 3.5A, lanes 15-16 and 3.5C, construct (ix)). 3.3.3 A6554 and A6576 contribute to IRES-mediated translation and reading frame selection  The PKI domain is comprised of a junction that contains two unpaired adenosines (A6554 and A6576) that may contribute to IRES structure and function. Previously, we observed a loss in 0 frame but not +1 frame translation upon deletion of A6554 (Figure 3.2B, blue box) (Ren et al., 2014). Substitution of A6554 to other bases did not yield significant defects in either 0 or +1 frame translation, and within two contexts (A6554C and A6554G), resulted in an enhancement in 0 frame translation (Figure 3.2B, blue box). These results indicate that the nucleotide identity at nucleotide position 6554 is not a determining factor in IRES-mediated reading frame selection. We next introduced base substitution or deletion at A6576 (Figure 3.2B, green box). Base substitution at A6576 affected IRES-mediated translation to varying extents, with 0 and +1 frame translation ranging from 83-115% and 60-139% of the wild-type IRES, respectively. Interestingly, deletion of A6576 severely inhibited translation in both 0 and +1 frames (3% and 22%, respectively). Thus, the presence of nucleotides 6554 and 6576 is required for IRES-mediated translation. We reasoned that the unpaired A's may affect the conformation of the SLIII/P3.3 helix relative to the P3.1/P3.2 helices. To address this, we introduced an additional adenosine residue independently at 6554 and 6576 and assessed translational activity (Figure 3.2B, InsA6554 and InsA6576). Insertion of an A at A6554 yielded an inverse effect to base deletion, where 0 frame translation was only moderately diminished (19% reduction) and +1 frame translation was severely 85  affected (65% reduction) (Figure 3.2B, blue box). A similar effect was observed upon insertion of an additional A at A6576, where +1 frame translation was essentially abolished and 0 frame translation was unaffected (Figure 3.2B, green box). Together, these results suggest that molecular interactions at the three-way helical junction involving the adenosine bulges at 6554 and 6576 are important for reading frame selection.  To further elucidate the cause of the translational defect associated with ΔA6576, a toeprinting assay was performed for the A6576 mutants (Figure 3.5B). Consistent with the observed translational activities for the base substitution mutants, no significant changes in toeprint intensities were observed for A6576C, A6576G, and A6576U (Figure 3.5D). Surprisingly, however, ΔA6576, which has negligible 0 and +1 frame translation, yielded a +14 toeprint with similar intensity to the wild-type IRES (Figure 3.5B, lane 12 and 3D). This result suggests that the translational defect of ΔA6576 is not due to impairment in ribosome positioning and may occur at a step downstream. 3.3.4 Structural probing analysis of mutant ΔA6554 IRES  Because deletion of A6554 results in exclusive +1 frame translation (Ren et al., 2014), we hypothesize that A6554 serves a crucial role in maintaining the structural integrity of the three-way junction. To address this, selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) assay was performed to interrogate the regional flexibility of nucleotides for mutant ΔA6554 IRES. The normalized SHAPE reactivities at each position were examined and mapped onto the secondary structure to identify nucleotides that are flexible relative to surrounding nucleotides (Figure 3.6). We superimposed the relative SHAPE reactivities of mutant ΔA6554 onto that of the wild-type IRES (Figure 3.7A). For both wild-type and ΔA6554 IRESs, SHAPE reactivities were primarily observed in the bases constituting the single-stranded variable loop region (VLR), the loop of SLIII and those near the termini of the PKI pseudoknot (Figures 3.6 and 3.7A), consistent with previous biochemical evidence suggesting that this domain is structurally dynamic (Jan and Sarnow, 2002; Nishiyama et al., 2003; Ren et al., 2014). 86     Figure 3.6. Summary of SHAPE analysis of the wild-type and ΔA6554 IRESs in solution. Normalized SHAPE reactivies (see Section 3.2.6) for each IRES RNA are derived from Figure 3.7A and are summarized on the corresponding secondary structure according to the legend indicated. SHAPE probing was performed by Qian Ren.    87    Figure 3.7  SHAPE probing analyses of wild-type and ΔA6554 IRESs in solution and bound to the ribosome. The difference in SHAPE modification profiles between the wild-type and ΔA6554 IRESs was determined in solution (A) and bound to the ribosome (B) (see Section 3.2.6). Normalized reactivities are shown as a function of the nucleotide position. The differences in normalized SHAPE reactivities between the mutant and wild-type IRESs are summarized on the secondary structure according to the legend indicated (right). Specific nucleotide positions are indicated for reference and major IRES structural elements are denoted. SHAPE probing was performed by Qian Ren.     88  Interestingly, for the ΔA6554 IRES mutant, the most prominent change in the SHAPE profile was an increase in reactivities of nucleotides A6576-G6584 along helix P3.3a and nucleotides U6570 and AG6573-4 along helix P3.2, suggesting inherent flexibility in this region or an alternate conformation (Figure 3.7A). We next monitored and compared the SHAPE reactivities of the mutant ΔA6554 IRES bound to the ribosome to that of the wild-type IRES-ribosome complex. Notably, we observed increased NMIA reactivities within P3.3a and the VLR in the mutant ΔA6554 IRES-ribosome complex (Figure 3.7B). In general, the overall structures of the mutant ΔA6554 IRES are similar both in solution and bound to the ribosome, consistent with previous findings that the IRES may adopt a conformation that is associated with reading frame selection prior to ribosome binding (Ren et al., 2014).  To determine if the increase in SHAPE reactivities is correlated with the loss of Watson-Crick base-pairing, dimethyl sulfate (DMS) probing was performed using the wild-type and mutant ΔA6554 IRESs to identify unpaired A and C residues. As expected, DMS-modified nucleotides in the wild-type IRES resided in single-stranded regions, including nucleotides within the SLIII apical loop and the VLR (Figure 3.8A). Although some subtle differences were observed, there is general agreement between SHAPE and DMS profiles for the wild-type IRES (Figure 3.6 and 3.8A). Interestingly, for the mutant ΔA6554 IRES, residues within P3.3a and P3.2 helices exhibited relatively little or no DMS reactivity despite high SHAPE reactivity (Figure 3.6 and 3.8B). Overall, while the local nucleotide flexibility may be enhanced, the Watson-Crick edges remain inaccessible to modification, thus suggesting that the P3.3 and P3.2 helices may be still intact in the ΔA6554 mutant or that this region adopts an alternate conformation. 3.3.5 Position of translocated ribosomes on the IAPV IRES   Mutational analysis suggests that the structural integrity of SLIII is important for reading frame selection, and in particular, for IRES-mediated translation initiation in the 0 frame (Figures 3.3 and 3.4). To identify the step at which reading frame selection occurs, we sought to reconstitute the initial 89  translocation events mediated by the IGR IRES. Previously, it was proposed that the IRES PKI domain docks into the P site upon initial binding to the ribosome. This model was supported by observations that ribosome positioning on the IRES yielded +13-14 or +15-16 nucleotide toeprints, suggestive of P-site occupancy by the IRES PKI domain (Jan et al., 2003; Jan and Sarnow, 2002; Pestova and Hellen, 2003; Wilson et al., 2000a). Furthermore, reconstitution of translation in the presence of the elongation inhibitor cycloheximide resulted in the relative movement of the ribosome by six nucleotides, indicating the occurrence of two cycles of elongation (Jan et al., 2003; Jan and Sarnow, 2002; Pestova and Hellen, 2003; Wilson et al., 2000a). Cycloheximide inhibits translation by binding to the ribosomal E site, and as such, these observations were consistent with a model where the IRES initially occupies the P site to direct translation from the A site (Garreau de Loubresse et al., 2014; Pestova and Hellen, 2003). In light of the recent high-resolution structural data presenting evidence that initial ribosome binding positions the PKI domain of the IRES in the ribosomal A site, there is a need to re-evaluate the initial IRES-mediated translocation steps and understand the role of cycloheximide during this process (Fernandez et al., 2014; Koh et al., 2014).   We previously showed that IRES-mediated +1 frame translation can be reconstituted using minimal factors (Ren et al., 2014). Specifically, using an IRES mutant that is deficient in 0 frame translation by mutating the first 0 frame GGC glycine codon to a UAG stop codon, and restoring +1 frame translation by a U6562G substitution (Figure 3.9, construct (x), U6562G/GGC6618-20UAG), we observed a +21-nucleotide toeprint in the presence of cycloheximide (7 nucleotides downstream of the +14-nucleotide positioning toeprint) which we interpreted as a ribosome that has undergone two translocation cycles in the +1 frame (Figure 3.9, lane 5). As expected, the +21 toeprint was not observed in the absence of cycloheximide (Figure 3.9, lane 6) (Ren et al., 2014). To identify the step at which reading frame is selected, we generated novel mutants harboring consecutive +1 frame stop codons (Figure 3.9, +1F S2 and +1F S3), which in effect circumvent the need for cycloheximide to stall the ribosome and allow direct monitoring of translocation events in the +1 frame. Replacing the +1 frame  90     Figure 3.8. DMS probing analyses of wild-type and ΔA6554 IRESs. DMS modification profiles of (A) wild-type and (B) ΔA6554 IRESs in solution (see Section 3.2.6). Normalized reactivities are shown as a function of the nucleotide position. DMS reactivities are summarized on the secondary structure according to the legend indicated (right). Specific nucleotide positions are indicated for reference and major IRES structural elements are denoted.     91  second codon to a stop codon (+1F S2) allows the ribosome to be stalled following translocation when the stop codon enters the A site. Based on the recent structures of IRES/ribosome complexes (Fernandez et al., 2014; Koh et al., 2014), we expected that two consecutive translocation events would occur in the +1 frame, allowing PKI to first transit from the A to the P site thus permitting delivery of the first aminoacyl-tRNA (Arg) to the first codon, followed by a second translocation step resulting in the stop codon in the A site. Indeed, in the presence or absence of cycloheximide treatment, a +21-nucleotide toeprint was observed, consistent with the occurrence of two elongation cycles in the +1 reading frame (Figure 3.9, lanes 8-9). Because we did not observe a difference in the +14 toeprint upon ribosome binding to the IRES (Ren et al., 2014) (Figure 3.5), the occurrence of the +21 toeprint indicates that the reading frame is selected at a step within the first two translocation events. To monitor a subsequent translocation event, translocation was reconstituted using an IRES reporter containing a +1 frame UAG as the third codon. In the absence of cycloheximide, a novel +24-25-nucleotide toeprint was observed, indicating that three translocation events had occurred (Figure 3.9, lane 12). Intriguingly, only a +21 nucleotide toeprint, equivalent to two translocation cycles, was noted in the presence of cycloheximide (Figure 3.9, lane 11). Previous interpretations of biochemical data suggested that cycloheximide inhibits IRES translation when a deacylated tRNA is bound in the E site (Pestova and Hellen, 2003). Given the recent structural data that cycloheximide binds to the E site where the acceptor end of a tRNA normally resides (Garreau de Loubresse et al., 2014), we propose an alternate interpretation in which cycloheximide induces a premature block in the initial steps of IRES translation. This is likely achieved by inhibiting the process of translocation, with the IAPV PKI domain positioned in the E site and the first aminoacyl-tRNA in the P site (Figure 3.10). Thus, occupancy of the IAPV tRNA-like PKI domain in the E site can still accommodate cycloheximide binding, leading to inhibition of ribosome translocation. In summary, the toeprinting profiles are consistent with the model wherein the IAPV PKI domain initially occupies the ribosomal A site, and subsequently transits into the P site (Figure 3.10) (Fernandez et al., 2014; Koh et al., 2014).  92    Figure 3.9. Reconstitution of IRES-mediated translation restricted to the +1 frame. Top. Bicistronic IRES RNAs are incubated with purified, salt-washed human ribosomes, in the presence or absence of yeast elongation factors, bulk aminoacyl-tRNAs and the translation inhibitor cycloheximide, as indicated (see Section 3.2.5). Ribosome positioning is monitored by primer extension analysis, and the resultant cDNA products are resolved by denaturing polyacrylamide gel electrophoresis. Sequencing reactions are shown on the left, with the position of the +1 nucleotide as denoted. The locations of major toeprints including A6628 (+14), A6635 (+21) and CA6638-9 (+24-25), are indicated on the right. Bottom.  Schematics of IRES mutants with the location of the major toeprints as shown.   93    Figure 3.10. Pathway of IGR IRES-mediated translation initiation. The IGR IRES can bind directly to 40S and 60S ribosomal subunits. Upon initial ribosome binding, the PKI domain is positioned within the A site ('pre-translocation state'). Eukaryotic elongation factor (eEF) 2 mediates pseudotranslocation of PKI to the P site ('post-translocation state'), which is an unstable state that is susceptible to back-translocation unless stabilized by delivery of the first aminoacyl-tRNA to the A site by eEF1A. eEF2 mediates a second pseudotranslocation event which results in movement of the first aminoacyl-tRNA and the PKI domain to the P and E sites, respectively. IRES-mediated translation subsequently proceeds by the canonical elongation pathway. The elongation inhibitor, cycloheximide (CHX), blocks IRES-mediated translation following two translocation events, with PKI in the E site. Adapted with permission from (Ruehle et al., 2015).  94  3.3.6 Readout of eukaryotic release factor 1-dependent toeprints of IRES/ribosome complexes    Although ribosomes that have translocated in the +1 frame can be detected on the IRES after two translocation events (Figure 3.9), it is unclear whether reading frame selection by the IRES occurs upon initial translocation of the PKI domain from the A to the P site prior to aminoacyl-tRNA delivery, or specifically by delivery of the first aminoacyl-tRNA. To address this, we used a modified reconstituted system containing eukaryotic release factor 1 (eRF1). eRF1 functions to stabilize post-translocated complexes and to prevent spontaneous back-translocation of the IRES PKI domain following a single translocation event of PKI from the A site to the P site (Jan et al., 2003; Muhs et al., 2015). Furthermore, the incorporation of eRF1 into our minimally reconstituted system circumvents the need for aminoacyl-tRNAs, thus allowing us to examine the initial translocation of the PKI domain from the A to P site. In ribosomes assembled on mutant IRESs that contain a stop codon, eRF1 recognizes and binds to stop codons in the A site in an eEF2-dependent manner, resulting in a +4 nucleotide shift in the toeprint (Jan et al., 2003; Muhs et al., 2015). To determine if the +4 nucleotide eRF1 toeprint can be recapitulated using the IAPV IRES, we utilized IRES constructs harboring stop codons in the first codon of the 0 or +1 frame (WT 0FS1 or WT +1FS1, respectively) (Figure 3.11). Since the eRF1 toeprint is dependent on the presence of a stop codon, the wild-type IRES lacking a stop codon yielded no detectable eRF1-dependent toeprint, as expected (Figure 3.11, lane 2). Conversely, the WT 0FS1 IRES generated a robust toeprint at +4 nt, similar to that observed previously for the Cricket paralysis virus IRES (Jan et al., 2003; Muhs et al., 2015) (Figure 3.11, lane 4). Although the wild-type IAPV IRES supports +1 frame translation, no eRF1 toeprint was observed for WT +1FS1, possibly due to the lower level of +1 frame expression compared to 0 frame translation (Figure 3.11, lane 6). Alternatively, the presence of eRF1 may be shifting the equilibrium towards selection of the 0 frame. To characterize reading frame selection, we took advantage of two previously characterized mutants: G6568C, which only supports 0 frame translation, and ΔU6569, which exhibits exclusive +1 frame translation that is approximately 3-fold higher than wild-type activity (Ren et al., 2014). We selected these mutants because they initiate translation exclusively  95   Figure 3.11. Reconstitution of translocation using eukaryotic release factor 1 (eRF1). (Top) Bicistronic IRES RNAs, as described in the text on the previous page, were incubated with purified human ribosomes, GTP and eukaryotic elongation factor 2, in the presence or absence of eRF1, as indicated. Ribosome positioning was monitored by primer extension analysis, and the resultant cDNA products were analyzed by denaturing polyacrylamide gel electrophoresis (as in Section 3.2.5). Sequencing reactions are shown on the left, with the position of the +1 nucleotide as denoted. The locations of the major toeprints, including A6628 (+14), A6632 (+4 shift) and A6634 (+6 shift), are indicated. (Bottom) Schematics of IRES mutants and the location of the major toeprints are shown.   96  within one reading frame, and at a level that is similar to or exceeding wild-type activity (Ren et al., 2014). Surprisingly, while only the G6568C mutant exhibits exclusive 0 frame translational activity, both G6568C and ΔU6569 yielded a +4 toeprint when a stop codon is present in the first 0 frame codon for both constructs (Figure 3.11, lanes 8 and 12). Furthermore, with a +1FS1 mutation, a novel +6-nucleotide toeprint was observed for both the G6568C and ΔU6569 mutants (Figure 3.11, lanes 10 and 14).  Given these results, we demonstrate that the eRF1-dependent toeprint profiles of the mutant IRESs differ from that of the wild-type IRES, and the nature of mutation does not affect the unique toeprints observed. These results suggest that these mutations may affect how accurately the IRES selects the translational reading frame.  3.3.7 Structural determination of the IAPV PKI domain   Our data suggest that the IAPV PKI domain may be dynamic and can adopt distinct conformations to mediate 0 and +1 frame translation. To investigate this in further detail, our collaborator, Dr. Samuel Butcher (University of Wisconsin-Madison) determined the structure of the IAPV IRES PKI domain using a 70-nucleotide construct that contains the entire base-paired region in the pseudoknot domain (Figure 3.12A). The global fold of the 70-nucleotide IAPV IRES PKI domain was determined using a hybrid NMR/SAXS approach (Burke et al., 2012) (For additional experimental details and supplemental data, refer to Appendix B). The resulting overall fold reveals that PKI resembles a tRNA (Figure 3.12B). The PKI domain contains three main helices that intersect at a three-way junction, which contains the two unpaired bases, A6554 and A6576. Overlaying the PKI structure with tRNAPhe shows that P3.3 (SLIII) is analogous to the acceptor arm of a tRNA and is coaxially stacked with P3.1, which forms the elbow of a tRNA (Figure 3.12B). Furthermore, P3.2 and PKI helices are coaxially stacked to mimic the anticodon stem of a tRNA. Therefore, in this view the PKI pseudoknot helix and loop appears more analogous to the anticodon helix and loop of tRNA rather than an anticodon-codon interaction, the latter of which must form directly downstream of PKI. The 3’ terminal nucleotide of PKI is disordered in the models, so the trajectory of the first codon downstream of PKI cannot be defined from our data.  The  97    Figure 3.12. Structure of the IAPV PKI domain. (A) Secondary structure of the PKI domain used for structural determination. (B) Left. Structural ensemble of the IAPV IRES, as determined by NMR/SAXS. The structural elements are colored as per the secondary structure in (A). Right. The averaged structure of the IAPV PKI domain (orange) is overlaid onto the structure of a Phe-tRNA (green). SLIII mimics the tRNA acceptor stem. Structural studies are performed by our collaborators in Dr. Samuel Butcher's lab.     98  VLR is also disordered in the ensemble models and is consistent with previous structural probing data showing that this region is dynamic (Ren et al., 2014). While structural studies of the PKI domain of the Type I IRES show anticodon-codon mimicry (Costantino et al., 2008), the PKI domain of the IAPV IRES is the first example to display mimicry of the entire tRNA L-shape.    Dr. Samuel Butcher performed docking of the IAPV PKI domain into the ribosome using the cryo-EM models of the CrPV IRESs bound in the A and P sites of the yeast and rabbit ribosomes, respectively (Fernandez et al., 2014; Muhs et al., 2015) (Figure 3.13A and B). Specifically, he docked the anticodon-codon part of the IAPV PKI domain with the PKI domain of the CrPV IGR IRES. Overlaying the IAPV PKI domain with the CrPV IRES in the A site shows that the domain can be accommodated within the ribosome (Figure 3.13A). When modeled in the P site, the IAPV PKI is positioned where a tRNA normally occupies and exhibits good overlap with a post-translocated CrPV IRES (Figure 3.13B and C) (Muhs et al., 2015). The majority of the domain is well accommodated in the P site but a small degree of steric clash is observed for the 3’ nucleotide, which is disordered in our structural models due to lack of restraints for this terminal nucleotide.  In contrast to the recent cryo-EM structure of the Type II TSV IGR IRES, where SLIII is stacked coaxially on PKI (Koh et al., 2014), our model positions SLIII of the IAPV IRES along the trajectory of the tRNA acceptor stem within the ribosomal A site of the large subunit (Figure 3.13). Overall, the structure suggests that the tRNA-like shape of the PKI domain of the IAPV IRES allows access to the tRNA binding sites. 3.4 Discussion  The L-shape conformation of tRNAs is central for interactions with specific components of the ribosomal A, P and E sites and concomitantly with the mRNA via anticodon:codon pairing to ensure maintenance of the reading frame. Similarly, the dicistrovirus IGR IRES adopts a conformation that occupies the ribosomal tRNA-binding sites in order to direct factorless translation initiation from a non-AUG start codon, thus setting the ribosome into an elongation mode (Costantino et al., 2008; Fernandez et al., 2014; Koh et al., 2014; Muhs et al., 2015; Schuler et al., 2006; Spahn et al., 2004a; Wilson et al., 2000a). Anticodon-codon mimicry enables the PKI domain to occupy the A site and subsequently the P  99    Figure 3.13. Docking of the IAPV PKI domain. (A) Left. The IAPV PKI domain (red) is superimposed onto the structure of the CrPV IGR IRES bound in the A site of the yeast ribosome (PDB ID 4V91, (Fernandez et al., 2014)). The CrPV IRES (yellow), large ribosomal subunit RNA (green), and small ribosomal subunit RNA (cyan) are shown. Right. Zoom in view showing that SLIII (red) can be accommodated into the large ribosomal subunit that is normally occupied by the acceptor stem of a ribosomal A site tRNA. (B) Left. The IAPV PKI domain (red) superimposed onto the structure of the CrPV IGR IRES bound in the P site of the O. cuniculus ribosome (PDB ID 4D5Y, (Muhs et al., 2015)).  Right. Zoom in view.  (C) The IAPV PKI domain (red) is superimposed onto the structure of the CrPV IGR IRES in the post-translocated state (yellow) (Muhs et al., 2015). Docking studies are performed by Dr. Samuel Butcher.    100  site in order to allow delivery of the first aminoacyl-tRNA and thereby establish the reading frame (Costantino et al., 2008; Fernandez et al., 2014; Koh et al., 2014; Muhs et al., 2015). In this study, we utilized an NMR/SAXS hybrid approach to obtain a structural model of the PKI domain of the IAPV IRES, revealing complete tRNA-mimicry where the SLIII structural element resembles the acceptor stem of a tRNA. Through a series of biochemical and mutagenesis analyses, we also showed that the integrity of the SLIII domain and the two unpaired adenosines at the core junction of the three helices of the PKI domain are important in adopting the optimal RNA conformation for IRES-mediated reading frame selection. Structural mimicry of a natural tRNA likely allows the IRES PKI domain to recapitulate interactions with the ribosome in order to facilitate translation initiation and direct reading frame selection.  In contrast to the recent cryo-EM structure of the Type II TSV IRES (Koh et al., 2014), our current model reveals that the SLIII element of the IAPV IRES resembles the trajectory of the tRNA acceptor stem (Figure 3.12). Deviations in the orientation of SLIII between our structure and that of the TSV IRES may be explained by the following. First, the structure of the TSV IRES was solved bound to the ribosome whereas our model is of the free IAPV PKI domain. In the cryo-EM structure (Koh et al., 2014), the density of SLIII is weak and incomplete in this region of the map, thus suggesting that SLIII may be dynamic. It is possible that the IAPV IRES may adopt a conformation similar to that observed with the TSV IRES when bound in the A site and then undergoes structural rearrangements to adopt the conformation of a complete tRNA upon translocation into the P site. Our SHAPE analysis of the wild-type and mutant IAPV IRESs suggests that the PKI domain is flexible and may adopt different conformations (Figure 3.6 and 3.7). Second, the TSV IRES does not support +1 frame translation and as such, may not sample the full range of conformational states that mediate alternative reading frame selection. Lastly, the longer length of the IAPV IRES SLIII (6 base-pairs for the TSV IRES versus 8 base-pairs for the IAPV IRES) may impose a constraint when bound to the ribosome that is different than that of the TSV IRES SLIII. It is noted that the difference in SLIII length is likely not the only contributing factor in reading frame selection within the context of the IRES.  101   The overall shape of the PKI domain nearly resembles the shape of a tRNA. The most notable difference is the shorter anticodon stem region (P3.2) of the IAPV PKI (Figure 3.12). It is possible that upon binding to the ribosome, the P3.2 region becomes extended, thus filling the space of the entire anticodon stem of a tRNA in the ribosomal P and A sites. However, overlay of the IAPV PKI domain with the CrPV IRES bound to the ribosome reveals that the acceptor stem (SLIII) of the IAPV PKI domain can fit within the space of a tRNA within the large ribosomal subunit (Figure 3.13).  tRNAs adopt a conformation that relies on a tertiary structural interaction between the D and T loops (Moras et al., 1980). Remarkably, the IAPV IRES PKI domain, which is comprised of only three helices that intersect at a three-way junction, resembles a complete tRNA: SLIII fully mimics the acceptor stem of a tRNA and helices P3.2 and PKI are continuously stacked to resemble the anticodon stem. At the junction, the two unpaired nucleotides, A6554 and A6576, are likely important in mediating the overall shape of PKI, notably the angle to which SLIII is oriented relative to the P3.2 helix. Previously, it has been shown that the topology of three-way junctions, such as the angle of helices, can be classified according to a set of rules based on the number and location of unpaired nucleotides at the junction (Lescoute and Westhof, 2006). From analysis of the IAPV PKI domain, the presence of A6554 and A6576 predictably fits within the classification of three-way junctions with the P3.2 helix bent towards the coaxially stacked P3.1 helix. We speculate that the two unpaired A's interact with each other in order to facilitate the tRNA-like conformation, although this cannot be fully substantiated by the current NMR/SAXS model of the PKI domain. However, our data point to an important role of the unpaired A's in IRES-mediated reading frame selection. Mutation of A6554 or A6576 to other bases did not have a significant effect on 0 or +1 frame translation (Figure 3.2) (Ren et al., 2014)), which suggests that the ribose or the phosphate backbone, rather than the identities of the two bases, may be important. It is worth noting that the two bulged nucleotides proximal to the three-way junction, though prevalent across Type II IGR IRESs, are not conserved in identity (Nakashima and Uchiumi, 2009). As such, the indiscriminate 102  identity of the bases at 6554 and 6576 may suggest that various types of base interactions can sufficiently mediate the optimal tRNA-like conformation.  An emerging theme from our structural probing data suggests that local structural rearrangements, possibly representing distinct conformations or conformational intermediates of the IAPV IRES, facilitate differential reading frame selection (Ren et al., 2014). For the ΔA6554 mutant IRES which showed exclusive +1 frame translation, only one strand of helix P3.3 (P3.3a) showed increased SHAPE reactivities, yet minimal to no reactivity to DMS (Figure 3.7 and 3.8). At first analysis, this result may suggest that the base-pairing is still intact and that the increased SHAPE reactivities may be suggestive of conformational dynamics of the ribose sugar puckers (Gherghe et al., 2008; Mortimer and Weeks, 2009). An alternative explanation is that the ΔA6554 PKI domain adopts an alternate conformation that is not productive in mediating 0 frame but still maintains +1 frame translation. Given that the IRES is conformationally dynamic and may adopt several conformations that are associated with 0 or +1 frame translation (Ren et al., 2014), specific mutations may shift the equilibrium to a conformation(s) leading to exclusive 0 or +1 frame translation. Thus, the increased NMIA reactivities of P3.3a within ΔA6554 may represent a conformation or a conformational intermediate that leads to exclusive +1 frame translation. Furthermore, specific mutations within the PKI domain may enhance the flexibility of the three-way junction, resulting in a loss of translational fidelity, that manifests as exclusive 0 or +1 frame translation. For instance, insertion of an extra A at 6554 or 6576 yielded a drastic defect in +1 frame translation (Figure 3.2). These effects may be reminiscent of the suppressor mutant tRNATrp that contains the A9C mutation, which is located distally to the anticodon and causes increased nonsense suppression (Schmeing et al., 2011). The A9C mutation destabilizes packing and hydrogen-bonding of a base-triple located at a helical junction of the tRNA, enhances flexibility and consequently facilitates distortion of the tRNA that is intrinsic to the decoding process (Schmeing et al., 2011). Although the A9C tRNATrp explains how increased flexibility of the tRNA allows access to the A/T hybrid state, reading frame selection by the 103  IAPV IRES likely occurs from the ribosomal P site. Further investigations are needed to resolve whether conformational dynamics of the IRES PKI domain contribute to reading frame selection.   Our mutagenesis analyses indicate that the structural integrity of SLIII is important for 0 frame, but not +1 frame translation (Figures 3.3 and 3.4). Mimicry of the acceptor stem of a tRNA probably enables the SLIII to interact with specific components of the ribosomal P site to direct 0 frame translation, similar to that of a natural tRNA interacting with the ribosomal core during elongation. These results also imply that interactions of SLIII with the ribosome are not required for, or that the loss of these interactions underlie +1 frame translation. The P-site tRNA interacts with several ribosomal proteins and both small and large rRNAs that may contribute to reading frame maintenance (Atkins and Bjork, 2009). One proposed model for reading frame maintenance is the "ribosomal grip" - the interaction of the ribosome with the peptidyl-tRNA - which prevents slippage of the reading frame during translation (Nasvall et al., 2009). Consistent with this, mutations in the C-terminal tail of rpS9, which directly contacts the peptidyl-tRNA, induce errors in reading frame maintenance (Nasvall et al., 2009), suggesting that interactions between the ribosome-bound tRNAs and specific ribosomal compartments contribute significantly to reading frame maintenance. Similarly, mutations in rpL5 can lead to increased -1 and +1 frameshifting (Llacer et al., 2015). Critical contacts between the tRNA-like PKI domain of the IAPV IRES and specific ribosomal components may be essential for reading frame selection and/or maintenance during IRES-mediated translation.  The current model of the initial steps of IGR IRES translation involves the sequential translocation of the PKI domain through the A, P, and E sites. Initial binding of the IRES to the ribosome places the PKI domain in the ribosomal A site (Fernandez et al., 2014; Koh et al., 2014), which must translocate to the P site to present the next codon in the A site to the incoming aminoacyl-tRNA. The translational reading frame selected by the IAPV IRES is ultimately dictated by the delivery of the first aminoacyl-tRNA - the 0 frame Gly-tRNAGly or the +1 frame Ala-tRNAAla - but reading frame selection may occur prior to this when PKI is docked in the A site, upon translocation from the A to P sites, or 104  when PKI occupies the P site. Our previous and current studies have identified specific mutants that uncouple 0 and +1 frame translation without an effect on ribosome positioning in IRES/ribosome complexes (Ren et al., 2014), suggesting that reading frame is established downstream of PKI binding in the A site. Interestingly, our SHAPE analysis of the wild-type and mutant IRESs indicates that the IRES conformations do not change significantly upon ribosome binding, thus supporting the idea that the IRES may adopt distinct conformations that are primed to direct translation in a specific reading frame (Ren et al., 2014). Differential reading frame selection may be due to disruption of key ribosome-IRES interactions that consequently alter the IRES' ability to accurately discriminate and select the translational reading frame. Alternatively, mutations that lead to exclusive IRES-mediated +1 frame translation may result in conformations that occlude the 0 frame aminoacyl-tRNA to allow delivery of the +1 frame aminoacyl-tRNA (Ren et al., 2014).   tRNA mimicry appears to be a common strategy to manipulate and hijack the ribosome. tRNA-mimicry is observed in transfer mRNA (tmRNA), which is involved in translation-coupled mRNA surveillance pathways, specifically in No-Go Decay and bacterial trans-translation (Barends et al., 2011). Similarly, tRNA-like structures have been found in the 3'UTRs of some plant viral RNAs (Colussi et al., 2014; Dreher, 2010). In both cases, the tRNA-like structures can be aminoacylated, bind to ribosomes and participate in translation. We now show that complete mimicry is important for a subset of dicistrovirus Type II IRESs to direct translation in two overlapping reading frames. Overall, we demonstrate that tRNA shape-mimicry is a viral IRES strategy to initiate factorless translation and is important for reading frame selection in order to increase the coding capacity of a viral genome.     105  CHAPTER 4: AN ADJACENT STEM-LOOP IN HONEY BEE DICISTROVIRUSES PROMOTES IRES-MEDIATED TRANSLATION AND VIRUS INFECTION 4.1 Introduction  Bioinformatic analysis is a powerful approach that can be applied to identify novel conserved features within the viral genome that may represent functional elements involved in viral translation and/or replication. Sequence alignment of the honey bee (IAPV, KBV, ABPV) and fire ant (Solenopsis invicta virus-1) dicistroviruses resulted in the discovery of a novel overlapping gene (Firth et al., 2009; Sabath et al., 2009). The alternate gene, ORFx, is encoded in the +1 translational reading frame within the 5' proximal region of the cistron that codes for viral structural proteins (Firth et al., 2009; Sabath et al., 2009). ORFx translation initiates via a U:G wobble base-pair adjacent to the IRES translation start site, and although its expression has been confirmed in virally-infected honey bees, its function remains under investigation (Ren et al., 2014). In the same study, a hairpin element, designated stem-loop (SL) VI, was identified 5'-adjacent to the IGR IRESs of IAPV, KBV, and ABPV (Firth et al., 2009). The stem-loop structures of IAPV and KBV are comprised of 18 consecutive base-pairs, with the termination codon of the first viral cistron positioned in the apical loop (Figure 4.1A and B). A similar but shorter stem-loop  (14 base-pairs) was identified upstream of the ABPV IGR IRES, but differs in the position of the termination codon, which is encompassed in the helical stem of SLVI. Reporter constructs have been used to demonstrate that SLVI acts cooperatively with IAPV ORF2 partial sequence (nucleotides 6618-6908) to elicit an approximate two-fold stimulatory effect on IGR IRES activity in vitro. However, within the context of a bicistronic reporter construct, fusion of SLVI in frame with the upstream reporter such that ORF1 is in frame with the stop codon within SLVI - thus mimicking the natural genomic arrangement of IAPV - yielded a consistent decrease in both cap- and IRES-dependent translation (Ren et al., 2012). Furthermore, disruption of the SLVI helical stem through independent 5' or 3' mutations diminished both IRES-mediated 0 and +1 frame translation (Ren et al., 2012). IRES activity was rescued upon restoration of the helical stem, thus suggesting that formation of an intact stem-loop contributes to optimal IRES-106  dependent translation (Ren et al., 2012). While the use of reporter constructs suggests that the functional contribution of SLVI is exerted primarily at the level of translation, it is plausible that SLVI may have alternate functions. Studies into the contribution of SLVI to other aspects of the viral life cycle have been hampered by the lack of an infectious clone of IAPV or other honey bee dicistroviruses. An infectious clone of the related Cricket paralysis virus (CrPV) has only been recently established (Kerr et al., 2015), and has proven to be an effective tool in elucidating the molecular mechanisms of pathogenesis and viral-host interactions. To circumvent this hurdle and to begin to elucidate the functional relevance of SLVI within the context of virus infection, we engineered a chimeric virus that is derived from the CrPV infectious clone. We used Drosophila S2 cells as a model system and demonstrate that this chimeric virus is infectious. Despite its heterologous nature, this system represents an invaluable model to glean novel insights into how the IAPV IRES function impacts virus infection - studies which were not achievable prior to the development of this chimeric infectious clone. 4.2 Materials and methods 4.2.1 Reporter constructs Monocistronic and bicistronic luciferase reporter constructs containing the wild-type or mutant IAPV IGR IRES have been described previously (Ren et al., 2012) (see Appendix A).  4.2.2 RNA structural probing RNA structural probing was performed as described in Section 3.2.6. 10 pmol of monocistronic RNA bearing the wild-type or mutant IRESs was briefly heated to 95°C, followed by slow cooling to 30°C for 20 minutes in Buffer E (20 mM Tris-Cl [pH 7.5], 100 mM KCl, 2.5 mM MgOAc, 0.25 mM spermidine) to induce folding. The pre-folded RNA was treated with 1:200 dilution (in 100% ethanol) of DMS for 10 minutes at 30°C, in the presence of nonspecific yeast tRNAs. Following incubation, the reaction was terminated by the addition of Quench Buffer (30% (v/v) β-mercaptoethanol, 300 mM NaOAc [pH 5.0]) (Tijerina et al., 2007) and the modified RNAs were recovered by ethanol precipitation. For DMS-107  modified RNAs, primer extension was performed using 5'-end labeled primer PrHA190 (5'-ACCAGAGTTTATTGGAAATTTCCTC-3') and reactions were subsequently analyzed by 8% (w/v) denaturing gel electrophoresis. Gels were subsequently dried and subjected to gel electrophoresis.  4.2.3 Purification of 40S and 60S ribosomal subunits Purification of 40S and 60S subunits was performed as described in Section 2.2.3. 40S and 60S ribosomal subunits were purified from HeLa pellets (National Cell Culture Center), as described previously (Jan and Sarnow, 2002). HeLa cells were lysed in lysis buffer (15 mM Tris-Cl [pH 7.5], 300 mM NaCl, 1% (v/v) Triton X-100, 6 mM MgCl2, 1 mg/mL heparin) and the supernatant was subjected to brief centrifugation to remove cellular debris. The supernatant was applied to a 30% (w/w) sucrose cushion containing 500 mM KCl and centrifuged at 100,000 g to pellet ribosomes. Ribosomes were resuspended in Buffer B (20 mM Tris-Cl [pH 7.5], 6 mM MgOAc, 150 mM KOAc, 6.8% (w/w) sucrose, 2 mM DTT) and subsequently treated with puromycin (2.3 mM final concentration) to dissociate ribosomes from the mRNAs. Potassium chloride was added to a final concentration of 500 mM. The dissociated ribosomes were resolved on a 10-30% (w/w) sucrose gradient where the peaks corresponding to the free 40S and 60S subunits were detected by measuring the absorbance at 260 nm. The corresponding fractions were collected, pooled, and concentrated in Buffer C (20 mM Tris-Cl [pH 7.5], 0.2 mM EDTA, 10 mM potassium chloride, 1 mM magnesium chloride, 6.8% (w/w) sucrose) using Amicon Ultra spin concentrations (Millipore). The concentrations of the ribosomal subunits were determined by spectrophotometry using the conversions 1 A260 nm = 50 nM and 1 A260 nm = 25 nM for the 40S and 60S subunits, respectively.  4.2.4 Toeprinting/ primer extension analysis Toeprinting analysis was performed as described in Section 3.2.4. 150 ng of bicistronic wild-type or mutant IRES RNAs were annealed to primer PrEJ761 (5'- CATGGGGGTATCGATCCTATTTGGAG-3') in 40 mM Tris-HCl (pH 7.5) and 0.2 mM EDTA by heating to 65°C, followed by slow cooling to 37°C. 108  The annealed RNAs were incubated with 100 nM and 150 nM final concentration of purified 40S and 60S subunits, respectively. Ribosome positioning was analyzed by primer extension/reverse transcription using 10 units of AMV reverse transcriptase (Promega) in the presence of 125 µM of each of deoxythymidine  triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate, 25 µM deoxyadenosine triphosphate, 0.5 µL of α- [32P] dATP (3.33 µM, 3000 Ci/mmol), 8 mM MgOAc, 10 units of Ribolock, 1X buffer E (20 mM Tris-HCl,(pH 7.5), 100 mM KCl, 2.5 mM MgOAc, 0.25 mM spermidine, 2 mM DTT) in the final reaction volume. Reverse transcription was performed at 37°C for 1 h, after which the reaction was quenched by extraction with phenol/chloroform (twice), chloroform (once) and ethanol precipitated. The resultant complementary DNA fragments were analyzed by 6% (w/v) denaturing gel electrophoresis. The gels were subsequently dried and subjected to phosphorimager analysis.  4.2.5 Construction of the CrPV/IAPV chimeric infectious clone The chimeric infectious clone was derived from the full-length CrPV infectious clone (pCrPV-3, (Kerr et al., 2015)) using the Gibson Assembly Master Mix (New England Biolabs), per the manufacturer's instructions. See Appendix A.  4.2.6 In vitro transcription and translation Purified plasmid of pCrPV-3 or the derived chimera was linearized using Ecl136II. RNA was transcribed in vitro using T7 RNA polymerase and purified using an RNeasy Kit (Qiagen). The purity and integrity of the RNA were confirmed by denaturing formaldehyde agarose gel electrophoresis. For in vitro translation, 3 µg of purified RNA was pre-folded in buffer E (20 mM Tris⋅HCl pH 7.5, 100 mM KOAc, 2.5 mM MgOAc, 0.25 mM spermidine, and 2 mM DTT) and incubated in a Spodoptera frugiperda (Sf21) extract (Promega) in the presence of [35S]-methionine/cysteine (Perkin-Elmer). The reactions were performed at 30°C for 2 h and  analyzed by SDS-PAGE. The gels were subsequently dried and subjected to autoradiography and phosphorimager analysis (Typhoon, Amersham).  109  4.2.7 Cell culture Drosophila Schneider line 2 (S2) cells were maintained and passaged at 25°C in Shields and Sang M3 insect media (Sigma-Aldrich) supplemented with 10% fetal bovine serum. 4.2.8 RNA transfection 3 µg of in vitro transcribed RNA derived from the previously linearized pCrPV-3 or chimeric clone was transfected into 3.0x106 S2 cells using Lipofectamine 2000 reagent (Life Technologies) per the manufacturer's instructions.  4.2.9 Virus infection Drosophila S2 cells are infected at the indicated multiplicity of infection in minimal PBS, with rocking at 25°C. After 30 min, cells are replenished with media and harvested at the indicated time point. For metabolic labeling, 250 µCi/mL of [ 35S]-methionine/cysteine (Perkin-Elmer) was added to cells 30 min prior to the end of the time point. Cells were washed once in 1xPBS and harvested in lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrapyrophosphate, 100 mM NaF, 17.5 mM β-glycerophosphate, protease inhibitor cocktail (Roche)).    4.2.10 Western blotting Cells were washed once using 1x phosphate-buffered saline (PBS) and harvested in lysis buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrapyrophosphate, 100 mM NaF, 17.5 mM β-glycerophosphate, protease inhibitor cocktail (Roche)), as described previously (Garrey et al., 2010). Equal amounts of protein lysate (10 µg) was resolved by SDS-PAGE and subsequently transferred onto polyvinylidene  difluoride Immobilon-FL membrane (Millipore). Following transfer, the membrane was blocked for 30 min at room temperature in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated overnight with rabbit polyclonal antibody raised against CrPV RdRp (1:5000) or CrPV VP2 (1:5000). Membranes were washed 3 times with TBST and 110  subsequently incubated with anti-rabbit IgG-horseradish peroxidase (1:20,000; GE Healthcare) for 1 h at room temperature.   4.2.11 Northern blotting Total RNA was isolated from S2 cells using TRIzol Reagent per the manufacturer's instructions. 5 µg of RNA was resolved by denaturing agarose gel electrophoresis and subsequently transferred onto Zeta-Probe blotting membrane (Bio-Rad). Radiolabeled probe complementary to the CrPV viral genome was synthesized using DecaLabel DNA Labeling Kit (Thermo Fisher Scientific) and hybridized to the membrane overnight. Radioactive bands were visualized by autoradiography and phosphorimager analysis (Typhoon, Amersham). 4.2.12 Viral titres At the indicated time point, S2 cells were washed once and harvested in 1x PBS by three freeze/thaw cycles. Naive cells were infected using serial dilutions of the supernatant for 30 min, and transferred to 96-well plate pretreated with Concanavalin A (0.5 mg/mL; MP Biomedicals). At 10 h p.i., cells were fixed with 3% paraformaldehyde and incubated in 1x PBS overnight. Cells were permeabilized by methanol treatment, incubated with anti-VP2 antibody (1:500 dilution in 0.05 mg/mL bovine serum albumin (BSA)) and subsequently with goat anti-rabbit Texas Red IgG (1:200 dilution in 0.5 mg/mL BSA; Life Technologies). Nuclei were stained using Hoechst dye (0.5µg/mL; Life Technologies). Infected cells were visualized and quantified using a Cellomics Arrayscan HCS instrument.     4.3 Results 4.3.1 Formation of SLVI can be disrupted by 5' or 3' mutations  We previously showed mutations that disrupt the helical stem of SLVI inhibited IRES activity in vitro (Figure 4.1D) (Ren et al., 2012). Because the functional contribution of SLVI has been investigated within two contexts - when it is fused in frame with the upstream reporter to reflect the genomic  111   Figure 4.1. Secondary structure of the IAPV IGR IRES and cognate stem-loop (SL) VI mutants. (A) The Dicistroviridae genome is comprised of a positive-sense, single-stranded RNA molecule that bears a 5' genome-linked viral protein (VPg) and a 3' poly(A) tail. The genome consists of two open reading frames (ORFs): the upstream cistron encodes viral non-structural proteins, and the downstream cistron encodes viral capsid proteins. ORF1 and 2 expression is translationally and temporally regulated by the 5' IRES and the IGR IRES, respectively. (B) The IGR IRES adopts a triple-pseudoknot structure consisting of three pseudoknots (PKs I, II, III), which fold independently into two domains. PKII/III together form the ribosome binding domain, while PKI forms the tRNA-mimicry domain to establish the translational reading frame. Stem-loop (SL) VI located 5'-adjacent to the IGR IRES (highlighted in grey) has been identified in a subset of dicistroviruses, including IAPV,  ABPV and KBV. (C) 5', 3' , and compensatory mutations in SLVI. The depicted secondary structures are predicted by Mfold. Mutated nucleotides are shown in white. For IAPV, the ORF1 stop codon is encompassed in the apical loop of SLVI (bolded in red). (D) In vitro translational activities of reporter RNAs harboring mutant SLVI IRESs, as published in (Ren et al., 2012).   112  arrangement, or as a discrete element in the intercistronic region of the dual luciferase reporter - the 5' mutation (M1) was designed such that the base substitutions result in synonymous changes upstream of the natural stop codon (Ren et al., 2012). The 3' mutation (M2) was designed to complement the 5' mutation, which, when introduced simultaneously, would restore the structure of the stem-loop (M1+2). To verify that such mutations conferred the intended effects in disruption of the helical stem, RNA structural probing was performed. Base-pair formation was interrogated using dimethyl sulfate (DMS), which methylates unpaired adenosines and cytosines at the N1 and N3 positions, respectively. Because the adduct impedes reverse transcriptase, the modification sites can be identified by primer extension analysis. In parallel, DMS probing was performed in the absence of DMS to control for arrest of the reverse transcriptase due to RNA structure.      For the wild-type IRES, treatment with DMS resulted in modification of the two adenosines within the apical loop of SLVI (Figure 4.2, lanes 1-2). Other adenosine and cytosine residues within the helical region did not exhibit reactivity to DMS, indicating that SLVI is likely formed as predicted by the secondary structural model. As control, the DMS profile was determined for a mutant IRES (ΔPKI-III) in which PKI/II/III base-pairing was disrupted. Although pseudoknot formation was impaired, DMS probing reveals that the modification profile of SLVI in the mutant resembles that of wild-type (Figure 4.2, lanes 3-4). For mutant M1, DMS modification was detected at unpaired adenosines and cytosines in the secondary structure prediction, including C6392, A6397, A6398, and A6401 (Figure 4.2, lanes 5-6). For mutant M2, DMS modification was observed at adenosine and cytosine residues which are not predicted to be involved in base-pairing, including A6397, A6398 and A6404 (Figure 4.2, lanes 7-8). While C6389 and A6410 are expected to be unpaired, DMS reactivity at these positions is low. Furthermore for both M1 and M2, reactivities were observed at some adenosines which have the potential to base-pair with adjacent nucleotides (A6385, A6388, A6391, A6393, A6403, A6413 for M1 and A6385, A6386, A6388, A6391, A6393 for M2), suggesting that these interactions may be absent or occurring transiently (Figure 4.2, lanes 5-8). Alternatively, it is possible that a subset of RNAs may be in an alternative conformation. 113  Compensatory mutation (M1+2) eliminated the additional reactivities observed for the mutant, suggesting that the helical stem structure is likely restored (Figure 4.2, lanes 9-10). Taken together, DMS probing results indicate that the 5' and 3' mutations are indeed disrupting the normal structure of SLVI, and when introduced concomitantly to restore base-pairing, SLVI formation is restored. 4.3.2 An intact SLVI contributes to proper positioning of the ribosome  Previously, we determined the translational activities of SLVI mutants in an Sf21 in vitro transcription-translation extract. Within the context of the bicistronic reporter construct, mutant M1 moderately decreased IRES-mediated translation, whereas mutant M2 inhibited IRES activity significantly. Restoration of SLVI formation by the M1+2 mutation rescued IRES-mediated translation (Figure 4.1D) (Ren et al., 2012). Preliminary filter binding performed by Valentina Elspass suggests that ribosome binding is not impaired for both M1 and M2 mutants (Appendix C). Given this observation, it is likely that the defect in translation occurs at a step downstream of ribosome binding. To determine if SLVI formation contributes to ribosome positioning, toeprinting/primer extension was performed on IRES/ribosome complexes. Reverse transcription terminates upon encountering the leading edge of the ribosome, and the resultant complementary DNA (the 'toeprint') can be used to infer the specific nucleotides of the query sequence that occupy the ribosomal A and P sites. Initial binding of the IRES to the ribosome positions the tRNA-like element in the A site (Fernandez et al., 2014; Koh et al., 2014); thus, toeprinting can help monitor A site occupancy by the PKI domain and consequently the selection of the appropriate reading frame. Toeprinting profiles were determined for bicistronic wild-type or mutant IAPV IRES RNAs bound to purified salt-washed ribosomes isolated from HeLa cells. Ribosome assembly on the wild-type IAPV IRES RNA yielded a discrete toeprint at A6628, 14 nucleotides downstream of the CCU codon constituting PKI base-pairing in the ribosomal A site, as observed previously (Figure 4.3A, lane 2 and Figure 4.3B) (Ren et al., 2014). As expected, disruption of PKI base-pairing (ΔPKI) or PKI/II/III interactions concomitantly (ΔPKI-III), eliminated the toeprint, consistent with the established  114    Figure 4.2. Dimethyl sulfate (DMS) probing of SLVI mutants. (A) Monocistronic RNAs bearing the wild-type or mutant IRESs are treated with 1:200 dilution of DMS, as described in Section 4.2.2. Modifications are analyzed by reverse transcription using γ-[32P]-ATP labeled primer, which terminates one nucleotide upstream to the site of modification. (B) DMS reactivities are summarized on the secondary structure. ▲ denotes residues that are modified, as expected; Δ denotes residues that are predicted to be base-paired but are susceptible to DMS modification; * denotes positions that are expected to be unpaired but exhibit low DMS reactivity.   115   role of PKI in ribosome positioning (Jan and Sarnow, 2002; Sasaki and Nakashima, 2000; Wilson et al., 2000a) (Figure 4.3A, lanes 4 and 6). For both the 5' and 3' SLVI mutants, the same A6628 toeprint was observed, albeit at somewhat reduced intensities (Figure 4.3A, lanes 8 and 10). The ability of ribosomes to assemble on mutant M2 was more severely impaired, as supported by the approximate 62% reduction in toeprint intensity (Figure 4.3C). A reproducible decrease in toeprint intensity was observed for M1 and M2, thus suggesting that the translational defect observed with SLVI disruption was due to impaired ribosome positioning (Figure 4.1D and 4.3). Additionally, restoration of stem-loop formation (M1+2) was sufficient to rescue the toeprint to approximately 82% of wild-type intensity (Figure 4.3A and C), further underscoring the importance of an intact SLVI in optimal ribosome positioning and IRES activity.        4.3.3 A CrPV/IAPV chimeric clone is infectious in Drosophila S2 cells  Having shown that impaired ribosome positioning is likely the culprit for the translational defects observed for SLVI mutants (Figure 4.3), we sought to investigate if and how these defects manifest during virus infection. Due to the lack of an infectious clone for IAPV or other honey bee dicistroviruses which would be amenable to genetic manipulation, we derived a chimeric virus from the related full-length CrPV infectious clone, pCrPV-3, by replacement of the CrPV IGR IRES with that of IAPV (Kerr et al., 2015). The substitution was made such that it included IAPV IRES sequences immediately downstream of SLVI and encompassing PKI base-pairing. The CrPV structural protein coding sequence follows downstream of the IAPV IRES (Figure 4.4A, -SLVI). To verify that mature viral proteins can be expressed and processed in vitro, in vitro transcribed RNA derived from the chimera was incubated in Spodoptera frugiperda (Sf21) translation extract in the presence of [35S]-methionine/cysteine. Protein expression was monitored alongside the wild-type CrPV RNA (pCrPV) for reference. As shown previously, pCrPV resulted in the expression of ORF1 and ORF2 viral proteins. For the chimera (-SLVI), proteins corresponding to the mature viral proteins were also detected, with a profile that reflected that of pCrPV (Figure 4.4B, lanes 2 and 4). To demonstrate specificity, a stop codon was introduced into ORF2 116    Figure 4.3. Disruption of SLVI formation impairs ribosome positioning on the IAPV IRES. (A) Wild-type or SLVI mutant IRES RNAs are incubated in the absence (-) or presence (+) of purified, salt-washed HeLa 80S ribosomes and analyzed by primer extension and denaturing polyacrylamide gel electrophoresis, as described in Section 4.2.4. The sequence of the wild-type IRES is shown on the left, with the position of the CCU triplet that occupies the A site as indicated (+1 denotes the first C). The positioning toeprint is observed at A6628, 14 nucleotides downstream. (B) Schematic of the IRES PKI domain, with the positioning toeprint as indicated. (C) Quantitation of the positioning toeprint observed for wild-type and mutant IRESs, calculated as a fraction of the total lane intensity and normalized to the wild-type IRES. Shown are the average toeprint intensities ± 1 s.d. from at least three independent experiments. Toeprinting/primer extension analysis was performed by Valentina Elspass.      117   (C6428T), which prevents the synthesis of ORF2 proteins (Kerr et al., 2015). Indeed, the pCrPV ORF2 STOP mutant eliminated the expression of the corresponding viral structural proteins, which was similarly observed for the chimera RNA (Figure 4.4B, lanes 3 and 5). Given these results, we demonstrate that viral nonstructural and structural proteins can be expressed in vitro from the chimeric clone.   To determine if the chimeric clone is infectious in cell culture, in vitro transcribed RNA was transfected into Drosophila S2 cells, and the expression of mature capsid protein VP2 was monitored by Western blotting at 48 hours post-transfection ( h p.t.) (Figure 4.4C). As expected, VP2 protein could be detected following transfection of pCrPV and similarly for chimera (-SLVI), suggesting that it is infectious in S2 cells. Consistent with this, decreased levels of tubulin were observed for both pCrPV and chimera (-SLVI) transfection, further supporting the onset of a productive infection which may be inducing cytopathic effects. To confirm that infectious virions are generated, cell lysates were collected and used to re-infect naive cells for viral titres. Results indicate that the chimera (-SLVI) is indeed infectious and the resultant titre is comparable to pCrPV (Figure 4.4D).   To address the role of SLVI during infection, the stem-loop was introduced into the chimera such that it is fused with ORF1 (+SLVIfused), or in the intercistronic region immediately downstream of the ORF1 termination codon (+SLVI). For both the (+SLVIfused) and (+SLVI) chimeras, incubation of the respective RNAs in an Sf21 extract yielded observable bands that corresponded to the mature viral structural proteins (Figure 4.5A). Surprisingly, however, expression of VP2 capsid protein was only detectable for the chimera (+SLVI), but not for the chimera (+SLVIfused) upon transfection of the RNA (Figure 4.5B). To determine if fusion of SLVI with the upstream cistron affects IRES activity specifically, Western blotting was performed using antibodies raised against the ORF1 viral RNA-dependent RNA polymerase (RdRp). While the expression of RdRp was observed for the chimeras (-SLVI) and (+SLVI), it was not detectable for chimera (+SLVIfused), suggesting impaired translation of both the upstream and downstream open reading frames (Figure 4.5B). To further confirm this, viral titres were performed using lysates collected at 48 h p.t. of chimera (+SLVIfused) RNA, which yielded no measurable infectious virions 118   Figure 4.4. The chimeric IAPV/CrPV virus is infectious in Drosophila S2 cells. (A) Schematic of the chimeric virus derived from the full-length CrPV infectious clone. (-SLVI): chimera lacking SLVI; (+SLVIfused): SLVI is fused in frame with ORF1 such that the stop codon (UAA) is encompassed in the apical loop; (+SLVI): SLVI resides in the intercistronic region, downstream of the ORF1 stop codon. (B) In vitro transcribed RNAs derived from the wild-type CrPV infectious clone (pCrPV), or the chimera lacking SLVI (chimera (-SLVI)) are incubated in Sf21 extract for 2 h at 30°C in the presence of [35S]-methionine/cysteine (see Section 4.2.6). A stop codon was introduced into ORF2 of each respective clone to demonstrate specificity of viral structural protein synthesis. The identities of the mature viral proteins, annotated based on predicted molecular weight, are indicated. (C) In vitro transcribed RNAs from pCrPV, chimera (-SLVI) or their corresponding ORF2 stop mutants were transfected into Drosophila S2 cells. At 48 h p.t., cells were harvested and the expression of  the VP2 viral capsid protein was monitored by Western blotting. (D) Viral titres measured at 48 h post-transfection of pCrPV and chimera (-SLV) RNAs. Shown are the average values from three independent experiments, ± 1 s.d.  119    Figure 4.5. Fusion of SLVI with the upstream cistron impairs structural protein synthesis. (A) In vitro translation profiles of the  chimera (-SLVI), (+SLVIfused) and (+SLVI) in Sf21 extract. (B) The expression of the viral RNA-dependent RNA polymerase (RdRp) and capsid protein (VP2) is monitored by Western blotting using cell lysates following transfection of chimera (-SLVI), (+SLVIfused) and (+SLVI) RNAs into Drosophila S2 cells (see Section 4.2.10). (C) Viral titres were measured at 48 h p.t. for chimera (-SLVI), (+SLVIfused), and (+SLVI) chimeric RNAs (see Section 4.2.12). Shown are the average values from three independent experiments, ± 1 s.d.    120  (Figure 4.5C). Given that the same chimera can be efficiently translated in vitro to produce both ORF1 and ORF2 proteins, we speculate that fusing SLVI with the upstream cistron, which adds 8 additional amino acids to the C-terminus of RdRp, may result in impairment of RdRp function and/or trigger its degradation.  4.3.4 Disruption of SLVI reduces viral yield due to suboptimal IRES translation  Because fusion of SLVI with the nonstructural ORF1 coding region prohibited virus infection, we resorted to using the chimera (+SLVI) to investigate the role of SLVI within the context of the viral genome. With the obvious caveat this genomic arrangement does not reflect that of the native virus, it will be difficult to assess the role, if any, of SLVI on viral replication; thus, we restricted our studies to specifically address how defects in IGR IRES-mediated translation affect the progression of the viral life cycle. To determine if the functional contribution of SLVI to IRES translation can be recapitulated in vitro using the infectious clone, we introduced identical 5', 3', and compensatory mutations into chimera (+SLVI) and monitored protein synthesis in an Sf21 extract. While the M1 mutation yielded robust viral protein translation similar to chimera (+SLVI), the M2 mutation exhibited substantially reduced translation of structural proteins, although nonstructural protein levels were unaffected. Restoration of SLVI structure rescued structural protein expression (Figure 4.6A).   To examine the kinetics of viral protein synthesis, S2 cells were infected using chimera (+SLVI) or the respective mutants at a multiplicity of infection (MOI) of 10. Translation of newly-synthesized viral proteins was monitored over time by [35S]-methionine/cysteine labeling, followed by SDS-PAGE and autoradiography. Mock-infected cells exhibited extensive incorporation of radioactive methionine/cysteine, indicating active translation (Figure 4.6B). At 4 hours post-infection (h p.i.), preferential translation of viral nonstructural and structural proteins was observed for chimera (+SLVI) and its cognate mutants, concomitant with the decrease in overall translation. While the translation profiles were similar for (+SLVI), M1 and M1+2 chimeras, mutant M2 exhibited an observable decrease 121  in viral structural protein levels, which reflected the in vitro activity of the mutant IRES (Figure 4.6B) (Ren et al., 2012). At 6 h p.i., diminished expression of structural proteins was still observed for M2, although the difference was less distinguishable. To further confirm this, immunoblotting using RdRp and VP2 antibodies was performed to detect the total levels of nonstructural and structural proteins. We observed decreased accumulation of VP2 protein for mutant M2 at 4 h p.i., despite no observable changes in RdRp levels for chimera (+SLVI) and its derivatives, consistent with the pulse-labeling profiles (Figure 4.6B). Although the activity of mutant M2 was significantly inhibited in vitro, as demonstrated by [35S]-methionine/cysteine pulse-labeling (Figure 4.6A), the accumulation of VP2 structural protein was detectable by Western blotting during infection (Figure 4.6B). This disparity may be due to active viral replication during infection, which results in an increase in viral RNA that can undergo translation.     Northern blotting did not reveal substantial changes in the accumulation of viral RNA between the mutant and wild-type chimeras (Figure 4.6C). These results altogether suggest that the perturbations in viral structural protein synthesis observed for mutant M2 are not due to impaired viral replication but can be attributed primarily to differences in IGR IRES activity. Finally, to determine how the translational defect associated with mutant M2 affects the output of infectious virions, cells were infected with the wild-type chimera (+SLVI) virus or the cognate M2 mutant virus at MOI of 1 or 10. Viral titres were measured using lysates collected at 6 h post-infection. For mutant M2, infection at MOI 10 yielded an approximate 50% decrease in viral titres compared to the wild-type chimera (Figure 4.6D). A similar reduction in the yield of infectious virions was also observed for infection at MOI 1; thus, our viral titre results indicate that suboptimal translation of viral structural proteins due to the 3' mutations of SLVI directly impacts viral infection. Overall, our results demonstrate that defects in IRES-mediated translation observed using reporter RNAs can be recapitulated within the context of the chimeric infectious clone.  4.4 Discussion    Some transcripts contain secondary structure that must be melted by the ribosome in order for translation elongation to proceed (Meyer and Miklos, 2005). For the honey bee dicistroviruses, IAPV, 122   Figure 4.6. Disruption of SLVI formation decreases viral yield. (A) In vitro transcribed RNAs derived from chimera (+SLVI) or its cognate mutants are incubated in Sf21 extract for 2 h at 30°C in the presence of [35S]-methionine/cysteine, as described in Section 4.2.6. Reactions were resolved by SDS-PAGE and visualized by autoradiography. The respective mutations are as indicated in Figure 4.1C. (B) Drosophila S2 cells are infected with chimera (+SLV) or its cognate mutants at MOI = 10. [35S]-methionine/cysteine was added 30 min prior to the indicated time points (2, 4, 6 h p.i.) to metabolically label newly-synthesized proteins (see Section 4.2.9). Labeled proteins were resolved by SDS-PAGE and subjected to phosphorimager analysis. The expression of viral RdRp and VP2 was monitored by Western blotting, as described in Section 4.2.10. (C) The accumulation of viral RNA was visualized by Northern blotting at 2, 4, and 6 h p.i. with chimera (+SLVI) and mutant M2 (MOI  = 10) (see Section 4.2.11). (D) Viral titres were measured following 6 h p.i. with chimera (+SLVI) and mutant M2 at MOI = 1 and 10, as described in Materials and Methods. 123  KBV and ABPV, the upstream cistron terminates within SLVI, which is presumably unwound by the ribosome to gain access to the stop codon. The peculiar location of SLVI 5'-adjacent to the intergenic region internal ribosome entry site prompted us to explore how the context of this structural element influences translation (Ren et al., 2012). The use of specifically designed reporter constructs enabled us to demonstrate that fusion of SLVI with the first cistron, similar to the natural genomic arrangement, decreases translation of both upstream and downstream open reading frames (Ren et al., 2012). Here, we derived a chimera from an infectious clone of the related Cricket paralysis virus to address if formation of SLVI is important for virus infection. We demonstrate that the chimera is infectious by transfection of the RNA into Drosophila S2 cells, which resulted in the expression of viral nonstructural and structural proteins and yielded virions that can re-infect naive cells. Importantly, disruption of SLVI through 3' mutations reduced viral protein synthesis by the IGR IRES due to an impairment in ribosome positioning, as supported by toeprinting/primer extension analysis. The defect in IRES-mediated translation resulted in a decrease in the viral titre. All together, our results demonstrate that the SLVI is an independent structural element that promotes IAPV IRES activity by facilitating proper ribosome positioning on the IRES.  While our results support the importance of SLVI integrity in optimal IRES activity, it is interesting to note that 5' and 3' mutations in SLVI yielded disparate effects on IRES-mediated translation. Consistent with in vitro reporter activities (Ren et al., 2012), the M2 chimera exhibited a greater defect in IRES translation, both in translation extracts and in infected cells. One potential explanation is that M1 and M2 mutations have differing effects on SLVI stability; however, comparison of the ΔG values of wild-type, M1, M2 and M1+2 chimeras suggest that there is no strong correlation of SLVI stability with the observed translational activities. Interestingly, structural probing results indicate that adenosine and cytosine residues which are capable of base-pairing with adjacent nucleotides are susceptible to DMS modification. This suggests that SLVI may be conformationally dynamic within the context of both M1 and M2 mutations or may be prone to misfolding. It is unclear why mutant M2 exhibits a greater defect in 124  IRES activity. It is plausible that M2 mutations may be disrupting specific interactions with the ribosome and/or an unknown interacting partner that decrease the efficiency of IRES-mediated translation, although compensatory mutation suggests that restoration of SLVI base-pairing is sufficient to rescue IRES activity. Alternatively, mutations introduced in M2 may indirectly affect the global conformation of the IGR IRES required for optimal expression of ORF2 proteins.   One major caveat with our current studies is that the chimeric infectious clone does not faithfully represent the actual genomic arrangement of IAPV, in which ORF1 is fused in frame with the SLVI stop codon. Because structural proteins are expressed from the same construct in vitro, fusion of SLVI at the 3' end of ORF1 likely renders the chimera not infectious by impairment of RdRp function, and not faulty expression, although this remains to be tested. Due to this limitation, the functional contribution of SLVI to viral RNA replication cannot be sufficiently addressed or discounted. It is possible that the defect associated with SLVI disruption is underestimated in the chimera; should SLVI contribute to replication or other aspects of the viral life cycle, the defect in infectivity may in fact be exacerbated. Despite the heterologous nature of this system, relevant insights can still be gleaned, and there are various historical examples of how heterologous models have served as useful platforms to dissect the viral life cycle. For example, early studies into the hepatitis C virus (HCV) life cycle have been severely hampered by the lack of an infectious clone and suitable small animal model, which has encouraged the development of genetically modified and liver chimeric mice that are permissive to HCV infection (Dorner et al., 2011; Mercer et al., 2001). Research tools for HCV have also expanded to include infectious pseudo-particles that are based on lentiviral or retroviral core particles displaying unmodified HCV glycoproteins (Bartosch et al., 2003; Hsu et al., 2003). Studies into the role of the dicistrovirus RNAi suppressor in modulating the Drosophila antiviral response were also possible using a recombinant Sindbis virus (Nayak et al., 2010). Thus, these heterologous systems have proven to been useful in recapitulating key steps in the viral life cycle, especially in cases where a bona fide infectious clone has not been established. RNA secondary structure may have regulatory roles in translation. For example, some 125  programmed ribosome frameshifts are dependent on cis-acting structured elements to facilitate displacement of the ribosome into an alternate reading frame (reviewed in (Gesteland and Atkins, 1996)). Hairpins or other secondary structures in the coding region represent a kinetic barrier that inevitably decreases the rate of elongation. The effect on translation rate at the decoding center is dependent on the GC content of RNA structures located 14 nucleotides downstream at the entry of the mRNA channel (Qu et al., 2011). Despite the presence of duplex structures, the ribosome has the capacity to unwind the mRNA and mediate strand separation (Takyar et al., 2005). The ribosome's intrinsic helicase activity is sufficient to unwind a synthetic inverted repeat of 150 nucleotides engineered into the coding region of mRNA, thus suggesting that extensive RNA structure does not arrest an elongating ribosome (Lingelbach and Dobberstein, 1988). Because our results suggest that formation of an intact SLVI is required for IGR IRES-mediated translation, its disruption during translation elongation likely reduces IRES activity. However, unwinding of the helical region may also induce sufficient pausing of the elongating ribosome to cause a pileup of ribosomes, thus resulting in a concomitant decrease in ORF1 expression (Ren et al., 2012). Indeed, this effect was observed in vitro in the translation of a construct where SLVI is fused with the upstream reporter, which lends support to a potential role of SLVI in mediating negative coupling between the two viral cistrons (Ren et al., 2012). It is provoking to consider that the different folded states of SLVI may facilitate temporal regulation of nonstructural and structural protein synthesis during infection. Early in infection, expression of ORF1 encoding the viral protease and replicase likely interferes with SLVI formation and consequently decreases IGR IRES-mediated translation. As infection progresses, the sensitivity of the 5'-UTR IRES to limiting factors may down-regulate ORF1 translation to allow SLVI to adopt its folded state. This may stimulate IGR IRES-mediated translation and enhance the expression of structural proteins for viral packaging. Temporal regulation of dicistrovirus infection is not unprecedented and has been observed during Cricket paralysis virus infection (Khong et al., 2016). Where SLVI is positioned in the IRES/ribosome complex is not known. Currently, there is no structure of the complete IAPV or a related honey bee dicistrovirus IGR IRES. However, given that the IGR IRES binds in the intersubunit space in the conserved core of the ribosome (Schuler et al., 2006; 126  Spahn et al., 2004a), it is plausible that SLVI may be interacting with specific ribosomal components to facilitate IRES-mediated translation. From the secondary structure of the IRES, SLVI is located adjacent to loop L1.1, which interacts with the L1 stalk of the E site within the IRES-80S complex (Pfingsten et al., 2006; Schuler et al., 2006; Spahn et al., 2004a). Recent cryo-EM studies suggest that this interaction couples dynamics of the small subunit with movement of the L1 stalk, which may facilitate translocation of the IRES (Muhs et al., 2015; Murray et al., 2016). Interestingly, mutations in L1.1 also resulted in impairment in ribosome positioning (Jang et al., 2009). Given its proximity to L1.1, we propose that SLVI likely interacts with ribosomal elements within the vicinity of the E site, and may have complementary roles to L1.1. The availability of a high-resolution structural model of the IAPV IGR IRES will be particularly advantageous in elucidating molecular interactions with the ribosome. Furthermore, SLVI is present in only a small subset of Type II IGR IRESs that are infectious to honey bees. This peculiarity hints at the possibility that this structural element may have a species-specific function, or may exert a more prominent effect on ribosomes of honey bee origin. Alternatively, SLVI may modulate a process that is intrinsic to the viral life cycle of honey bee dicistroviruses.   The functional role of SLVI in virus infection has remained elusive due to the lack of an infectious clone of IAPV. Here, the development of a chimeric clone enabled studies into the role of this structural element  in IAPV IRES-mediated translation and virus infection. This chimera will likely serve as an invaluable tool until a bona fide infectious clone of IAPV or another honey bee virus has been established.    127  CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS  The Dicistroviridae IGR IRES provides an extraordinary example of molecular mimicry. By adopting a structural conformation that mimics an authentic anticodon:codon interaction (Costantino et al., 2008), the IRES is able to bind to the conserved core of the ribosome. Remarkably, direct recruitment and positioning of the ribosome at the translational start site occurs in the absence of canonical initiation factors and initiator Met-tRNAi via what is perhaps the most streamlined mechanism of translation initiation (Jan and Sarnow, 2002; Pestova and Hellen, 2003; Sasaki and Nakashima, 1999, 2000; Wilson et al., 2000a). Upon initial ribosome binding, the PKI domain is positioned in the decoding center and must undergo a pseudotranslocation step to vacate the A site for delivery of the first aminoacyl-tRNA (Fernandez et al., 2014; Koh et al., 2014). Within the P site, the IRES achieves a conformational state that resembles a P/E hybrid state tRNA (Costantino et al., 2008). By circumventing the requirements for initiation factors and harnessing a key step in the elongation cycle, the IRES can evade regulatory mechanisms that are exerted at the level of translation initiation. The IRES can, therefore, effectively co-opt the host translational machinery to support efficient synthesis of viral proteins under infection.   During eukaryotic translation initiation, tRNA-mRNA anticodon:codon interaction in the P site specifies reading frame selection. Because the IGR IRES bypasses the requirement for initiation factors and initiator Met-tRNAi, the mechanism by which reading frame is established during IRES-mediated translation remains an outstanding question. To begin elucidating this, we utilized a series of biochemical assays to assess how disruption of specific structural elements within the IGR IRES influences reading frame selection. While this thesis primarily focuses on the mechanism of IGR IRES-mediated translation initiation, the insights that have been generated have widespread implications on processes that are fundamental to canonical translation, including translational reading frame selection and maintenance. In Chapter 2, the contribution of PKI constituents to CrPV IRES-mediated translation was explored. Specifically, the roles of the PKI helical stem, anticodon:codon-like base-pairing, and the variable loop region (VLR) were investigated using in vitro translation and toeprinting/primer extension assays. 128  Extensive mutagenesis of these structural elements suggests that the PKI domain is likely optimized for IRES-mediated translation. Our data reveal that the VLR connecting the anticodon- and codon-like elements in cis is flexible and can mediate initiation from an adjacent alternate start site, although there is an inherent preference for the authentic site. Consistent with this, we demonstrate that nucleotide insertions can be tolerated in the VLR but at a detriment to IRES activity. Recent data hint at a possible role of the VLR in facilitating pseudotranslocation of the IRES (Ruehle et al., 2015). It will be of interest to identify interacting partners of the VLR, and to determine how such interactions may alter the conformation of the IRES/ribosome complex to facilitate translation. Furthermore, it will be interesting to explore how these mutations may affect viral infectivity using the full-length infectious clone of CrPV.    In light of the discovery that the a subset of dicistroviruses can mediate alternate reading frame selection using a novel, IRES-dependent mechanism (Ren et al., 2012), we adopted the use of the Israeli acute paralysis virus (IAPV) IGR IRES as a model to further elucidate the mechanism of IRES-mediated reading frame selection. In Chapter 3, we investigated the role of stem-loop III (SLIII), which is a unique structural feature within the PKI domain that is characteristic of Type II IGR IRESs. We demonstrate that disruption of SLIII integrity effectively uncoupled IRES-mediated 0 and +1 frame translation, and that 0 frame translation was adversely affected by shortening of the SLIII helical stem. Because SLIII structurally mimics the acceptor stem of tRNA, shortening SLIII may disrupt key interactions with the ribosome that are critical for reading frame maintenance. To determine if the reading frame is established upon initial ribosome binding, we examined the toeprinting profiles of a subset of SLIII deletion mutants and reveal that toeprint positions are unchanged in mutants that support 0 or +1 frame translation exclusively. Consistent with this, our reconstitution assays suggest that the reading frame is not established during the first pseudotranslocation event whereby PKI is displaced from the A site to the P site, but only upon delivery of the first 0 or +1 frame aminoacyl-tRNA.  This is in line with recent kinetic studies demonstrating that tRNA binding to the IRES/ribosome complex results in commitment to a specific reading frame (Petrov et al., 2016).  129   Previous work in our laboratory and the experimental data presented herein have provided recurring evidence suggesting that local structural rearrangements of the IRES may facilitate differential reading frame selection. For example, structural probing of the ΔA6554 mutant IRES, which showed exclusive +1 frame translation, exhibited enhanced SHAPE reactivities along SLIII, which is not due to the loss of Watson-Crick base-pairing. Since the IRES is conformationally dynamic, it may be plausible that specific mutation(s) (ie. ΔA6554) causes the IRES to adopt an alternative conformation that is incompetent in supporting differential reading frame selection. To gain structural insight, our collaborator, Dr. Samuel Butcher (University of Wisconsin-Madison), solved the structure of the IAPV PKI domain using NMR/SAXS hybrid approach (Burke et al., 2012). Remarkably, the overall conformation of the IAPV PKI domain exhibits striking resemblance to the L-shape of a complete tRNA. The three helices of the PKI domain intersect at a three-way junction that contains two unpaired adenosines, A6554 and A6576. SLIII fully mimics the trajectory of the tRNA acceptor stem, and helices P3.2 and PKI are continuously stacked to resemble the anticodon stem. Mutagenesis of A6554 and A6576 suggests that the two nucleotides play an essential role in IRES-mediated reading frame selection, possibly by maintaining the conformation and topology of the three-way junction. It will be interesting to explore how deletion of these adenosine bulges, particularly A6554, alters the structure and tRNA-like conformation of the PKI domain. Because the IAPV PKI domain mimics a complete tRNA, one prevailing theory is that such mutations may disrupt key interactions of the IRES with the ribosome that are implicated in reading frame maintenance. Structural and kinetic studies with these mutant IRESs will be helpful in obtaining mechanistic insights into reading frame selection.    In Chapter 4, we explored how IAPV IRES-mediated translational regulation affects the viral life cycle. Specifically, we investigated the function of the stem-loop element (SLVI) that is 5'-adjacent to the IGR IRESs of the honey bee dicistroviruses. Due to the lack of an IAPV infectious clone, we derived a chimeric virus from the full-length molecular clone of CrPV and monitored virus infection in Drosophila S2 cells (Kerr et al., 2015). Using this tool, we demonstrate that the position of the stem-loop relative to 130  the stop codon was critical and that mutations affecting stem-loop structure can lead to impaired IRES function. While this chimeric virus serves as a preliminary tool in understanding how IAPV IRES-mediated translation contributes to virus infection, there are significant limitations with its use. Firstly, the chimeric virus does not directly reflect the natural arrangement of the viral genome, where the ORF1 sequence is fused in frame with the stop codon of SLVI. Secondly, the chimeric virus contains only the minimal IAPV IGR IRES and does not encode the +1 frame ORFx sequence downstream of the IRES, thus limiting studies into differential reading frame selection and the function of ORFx. Thirdly, given the heterologous nature of the system, there is the inevitable caveat that any findings may not be representative of the biological processes that occur within the natural host. Thus, there is a clear need to establish a bona fide infectious clone of a honey bee dicistrovirus and a permissive cell line, which will likely be key in elucidating the molecular mechanisms of pathogenesis and viral-host interactions.   Structural and biochemical studies primarily featuring Type I IGR IRESs have substantially advanced our understanding of the mechanism of IGR IRES-mediated translation. It is likely that efforts are underway to obtain a high-resolution structural model of IAPV or a related honey bee dicistrovirus, which will allow us to derive structure-based mechanistic insights into alternate reading frame selection. Furthermore, the focus in the field will likely continue to shift towards the biology of dicistroviruses, and understanding how IRES-mediated translational regulation contributes to virus infection and pathogenesis. Since the recent establishment of the full-length Cricket paralysis virus infectious clone, considerable strides have already been made towards understanding the biology of virus infection (Kerr et al., 2015; Khong et al., 2016). The continued efforts in establishing a honey bee dicistrovirus infectious clone and permissive cell line will be significant and will provide a platform to elucidate fundamental processes in the viral life cycle of honey bee dicistroviruses and - importantly - shed light on the elusive function of ORFx.    Despite the unique strategy utilized by the IGR IRES in recruiting and positioning the ribosome, studies into its translational mode have broader implications for processes fundamental to translation. The 131  IGR IRES mimics a tRNA to directly bind to and manipulate the ribosome into an elongation mode. Thus, any relevant insights into how specific IRES-ribosome contacts promote conformational changes that are crucial for function can be generally extended towards understanding the dynamic roles of tRNAs and tRNA-mediated interactions in translation. Furthermore, binding of the IGR and HCV IRESs induces similar structural rearrangements in the ribosome (Spahn et al., 2004a; Spahn et al., 2001), suggesting that these conformational changes may be intrinsic to the translation process. Because the IGR IRES boasts a factor-independent translational mechanism, it can be harnessed for therapeutic uses to drive the co-expression of proteins for gene therapy. Historically, the encephalomyocarditis virus (EMCV) IRES has been utilized for this purpose; however, because the IGR IRES is refractory to many stress conditions and functions independently of translation factors, it may be a superior candidate with more widespread applications in therapy.    Our structure of the IAPV PKI domain provides a notable example of tRNA-mimicry that is achieved through non-canonical interactions. The tertiary structure of tRNA is dependent on long-range interactions between the D and TΨC arms. Despite significant deviations from the cloverleaf structure of tRNA, the IAPV IGR IRES is capable of adopting an overall similar topology: the anticodon stem is formed by the coaxially stacked PKI and helix P3.2; the unpaired adenosines (A6554 and A6576) intersect the helical junction to constitute the elbow region; and SLIII fully mimics the acceptor stem of tRNA. This remarkable molecular mimicry is not exclusive to IGR IRESs, but is exploited by other RNAs that encode regulatory elements. One prominent example is transfer-messenger RNA (tmRNA) and small protein B (SmpB), which mediate prokaryotic trans-translation to rescue stalled ribosomes on aberrant mRNAs. tmRNA and SmpB together recapitulate the canonical L-shape of tRNA (Bessho et al., 2007; Gutmann et al., 2003; Komine et al., 1994). During trans-translation, the tRNA-like structure (TLS) of tmRNA, which is aminoacylated, recognizes and enters the decoding center of a ribosome arrested on non-stop mRNA. Template switching allows translation to resume on the mRNA portion of tmRNA, which encodes a tag to target the aberrant protein for proteolytic degradation. Thus, the bifunctional 132  nature of tmRNA - which has both tRNA and mRNA activities - lies at the heart of this surveillance mechanism. Another striking example of tRNA mimicry can be also observed at the 3' termini of some plant viruses. These tRNA-like structures can be specifically aminoacylated and can form functional complexes with elongation factors (reviewed in (Dreher, 2009; Dreher and Miller, 2006)). The TLS of Turnip Yellow Mosaic Virus utilizes intramolecular interactions that are not canonically present in tRNA to achieve a similar topology. The overall tertiary fold is stabilized by a 'linchpin' interaction involving nucleotides that are downstream of a 5' pseudoknot element (Colussi et al., 2014). It is thought that formation or disruption of this interaction promotes different conformational states that may drive viral processes (Colussi et al., 2014). The examples of these diverse tRNA mimics thus illustrate the crucial role of divergent, non-canonical interactions in establishing a classic tRNA L-shape.  Given the remarkable tRNA-mimicry exemplified by the IAPV IGR IRES, it is intriguing to consider that the IRES may be subject to similar post-transcriptional modifications that may modulate its activity. In tRNA, position 34, which reads the wobble position of a codon, is enriched in modifications that influence codon recognition (reviewed in (Ranjan and Rodnina, 2016)) and modification of some tRNAs at this position fine-tunes the expression of specific genes (Rezgui et al., 2013). There is also emerging evidence that dynamic RNA modifications such as N6-methyladenosine are pervasive in messenger RNAs and function in various aspects of RNA metabolism (reviewed in (Meyer and Jaffrey, 2014; Yue et al., 2015)). N6-methyladenosine has also been identified in some viral genomes, and recent studies have described the role of this modification in modulating HIV mRNA expression (Kennedy et al., 2016; Tirumuru et al., 2016). The idea that the dicistrovirus genome and/or the IGR IRES is subject to post-transcriptional modifications that may modulate viral gene expression is provoking and warrants further investigation.  The RNA world hypothesis posits that RNA formed the basis of primordial life, owing to its ability to mediate catalysis and undergo replication. Given the streamlined nature of IRES-mediated translation, it is intriguing to consider that translation initiation in primitive eukaryotic life may have 133  occurred through an IRES-dependent mechanism. The view that IRESs may be remnants of the past can be justified by several considerations: firstly, the ability of IGR IRES to directly bind ribosomes suggests that specific RNA structures may have an inherent affinity for the ribosome; secondly, some modes of IRES-mediated translation can proceed in the absence of initiation factors and thus may be regarded as a more 'simplistic' process; thirdly, the 5' cap of mRNAs is an intrinsic requirement for cap-dependent translation, which arises from the catalytic function of protein factors (Hernandez, 2008). The strict dependence on protein activities during cap-dependent translation deems it an unlikely mechanism utilized by early life. It is plausible that a primitive mode of IRES-dependent mechanisms may have evolved earlier but was superseded by cap-dependent translation initiation as it offers more facets for regulatory control. However, because some viral IRES elements are still dependent on the activities of canonical factors, it is likely that they evolved from their hosts after the establishment of cap-dependent translation. Divergent evolution may have subsequently spawned more streamlined mechanisms of viral IRES-dependent translation due to specific selective pressures. In support of this, the non-essential ribosomal protein S25 is required for efficient translation of viral and cellular IRESs that utilize divergent strategies, suggesting that there may be a more universal mechanism governing IRES-dependent translation (Hertz et al., 2013; Landry et al., 2009; Muhs et al., 2011).   The evolutionary origin of the IGR IRES remains elusive. The striking tRNA-mimicry of IGR IRESs presents the enticing possibility that dicistroviruses may have acquired remnants of tRNAs or tRNA-like elements from their hosts. An alternate hypothesis is that the IGR IRES may have emerged as a byproduct of recombination events, yielding a chimeric species that has both ribosome binding and tRNA-like functionalities. Indeed, the two domains of IGR IRESs are modular and functionally independent, suggesting that they may have disparate origins (Hertz and Thompson, 2011; Jang and Jan, 2010). Recombination in RNA viruses, though occurring at variable rates, may perpetuate the emergence of genetically advantageous species and thus be of evolutionary significance (Simon-Loriere and Holmes, 2011). tRNAs serve as the critical link in deciphering the genetic information of mRNA into the 134  corresponding amino acid sequence. Their classic L-shape grants access to the ribosome's functional core and it is presumable that other RNA elements, similar to the IGR IRES, may partially or fully exploit this global conformation to do the same. 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Region of Mutation Construct Name Mutation CrPV IGR IRES PKI domain ProtRNA Substitution with anticodon stem-loop of prolyl-tRNA   ProLoop Substitution with anticodon loop of prolyl-tRNA    ProLoop1 Substitution with anticodon stem of prolyl-tRNA   ProLoop2 Substitution with anticodon stem of prolyl-tRNA/U6185G  PKI base-pairing ΔPKI    UA6190-1AU    UA6212-3AU    Comp   PKI helical stem ΔA6182    ΔU6176    ΔUU6176-7    ΔU6176/A6200    ΔUU6176-7/ AA6199-6200   VLR Ins1 Insert A following U6211   Ins2 Insert AC following U6211   Ins4 Insert ACGU following U6211   Ins7 Insert ACGUACG following U6211   Ins10 Insert ACGUACGUAC following U6211   Ins12 Insert ACGUACGUACGU following U6211   ΔU6203    ΔUA6203-4    ΔA6209    ΔAU6209-10    A6205C    A6205G    A6205U    A6207C    A6207G    A6207U    A6208C    A6208G    A6208U    A6211C    A6211G    A6211U   PKI 2ndsite Insert UACCU after C6218   2ndsite/CC6214-5GG  CC6214-5GG / Insert UACCU after C6218   2ndsite Adj Insert ACCU after U6216   2ndsite Adj/  CC6214-5GG CC6214-5GG / Insert ACCU after U6216 IAPV IGR IRES PKI domain UAA6586-8GGG    AAC6587-9CCU    ΔA6554    InsA6554 Insert A after A6554   A6576U    A6576C  150    A6576G    ΔA6576    InsA6576 Insert A after A6576  SLIII ΔC6579/ΔG6596    ΔG6580/ΔC6595    ΔU6581/ΔG6594    ΔA6582/ΔU6593    ΔCG6579-80/ ΔCG6595-6    ΔUA6581-2/ ΔUG6593-4    ΔAU6582-3/ ΔAU6592-3    ΔUCG6578-80/ ΔCGG6595-7    ΔUAU6581-3/ ΔAUG6592-4    UGC6593-5AUG    GUA6580-2CAU    GUA6580-2CAU/ UGC6593-5AUG    UGC6593-5UAU    GUA6580-2CAU/ UGC6593-5AUG    GCG6594-6UGC    CGU6579-81GCA    CGU6579-81GCA/ GCG6594-6UGC    CGU6579-81GCA/ GCG6594-6ACA    GCG6594-6ACA   Downstream of IGR IRES GGC6618-20UAG    construct (x) U6562G/GGC6618-20UAG   +1F S2 U6562G/GGC6618-20UAG/ AUU6622-4UAG   +1F S3 U6562G/GGC6618-20UAG/ CAC6625-7UAG   WT/0F S1 GGC6618-20UAG   WT/+1F S1 GCG6619-21UAG   G6568C/0F S1 G6568C/GGC6618-20UAG   G6568C/+1F S1 G6568C/GCG6619-21UAG   ΔU6569/0F S1 ΔU6569/GGC6618-20UAG   ΔU6569/+1F S1 ΔU6569/GCG6619-21UAG  SLVI M1 G6377C/U6380C/G6383U/A6386G/C6389U/ U6392C   M2 A6401G/G6404A/U6410A/A6413G/C6416G   M1+2 G6377C/U6380C/G6383U/A6386G/C6389U/ U6392C/ A6401G/G6404A/U6410A/A6413G/ C6416G pCrPV-3  ORF2STOP C6428T    151    Figure A.2. Plasmid map of the pCrPV-3 construct.  152  APPENDIX B: STRUCTURAL DETERMINATION OF IAPV PKI DOMAIN BY NMR/SAXS  (Performed by Dr. Samuel Butcher, University of Wisconsin-Madison)  MATERIALS AND METHODS RNA sample preparation for NMR RNA was transcribed in vitro using purified His6-tagged T7 RNA polymerase. DNA templates were purchased from Integrated DNA Technologies (IDT). 13C-15N labeled samples of PKI and PKIΔ6604-6618 were prepared using 13C-15N labeled nucleotides (Cambridge Isotope Laboratories). RNA samples were purified using denaturing 7.5-12.5% PAGE with 8 M urea. Impurities were removed by DEAE anion exchange (Bio-rad) using a low-salt buffer (20 mM Tris-HCl at pH 7.6, 200 mM sodium chloride) to wash and a high-salt buffer (20 mM Tris-HCl at pH 7.6, 1.5 M sodium chloride) to elute the RNA. Samples were then diluted to between 1-10 μM in 20 mM potassium phosphate (pH 6.3), 200 mM KCl, 0.5 μM EDTA, denatured for 5 min in boiling water, and annealed on ice for 30 minutes. RNA samples were concentrated to 0.5 mM in 300 μL with 10K molecular weight cut off centrifugal filter units (Millipore) at 4 °C. SAXS samples were subject to an additional size exclusion purification step using a HiLoad 16/500 Superdex 75 column (Amersham Biosciences) and dialyzed against 10 mM Tris (pH 6.3), 200 mM KCl, and 0.5 μM EDTA. All samples were assayed for folding homogeneity by 6% non-denaturing PAGE.  NMR data collection All spectra were obtained on Bruker Avance or Varian Inova spectrometers equipped with cryogenic single z-axis gradient HCN probes at the National Magnetic Resonance Facility at Madison. Imino resonances were assigned using 2D NOESY with a mixing time of 100 msec and 1H-15N 2D HMQC experiments at 10 °C. Partial alignment for RDC experiments was achieved by addition of 12.5 mg/mL Pf1 filamentous bacteriophage (ASLA) to a 13C, 15N U- and G-labeled sample. Pf1 phage concentration was confirmed by measuring 2H splitting at 700 MHz. Imino 1H-15N RDC measurements were obtained using 1H-15N 2D HMQC, 1H-15N 2D TROSY HSQC, and 1H-15N 2D Semi-TROSY HSQC experiments.  153  SAXS data collection  All SAXS data were obtained at Sector 12-ID-B and 5-ID-D of the Advanced Photon Source at Argonne National Laboratory. Measurements were carried out in 10 mM Tris (pH 6.3), 200 mM KCl, and 0.5 μM EDTA. RNA samples were loaded into a 1-mm capillary and flowed back and forth throughout the exposure. Twenty data collections of 0.5 sec each were averaged for each sample and buffer. The scattering intensity was obtained by subtracting the background scattering from the sample scattering. Subtraction of wide-angle scattering (WAXS) was adjusted until the contribution from buffer scattering was negligible. The scattering intensity at q = 0 Å-1 [I(0)], as determined by Guinier analysis, was compared between four different concentrations (0.5, 1.0, 1.5, and 2.0 mg/mL). WAXS and SAXS data were merged using the region between q = 0.09 Å-1 and 0.17 Å-1 in PRIMUS. Samples were assayed for radiation damage by denaturing 10% PAGE after data collection. No radiation damage was detected. Molecular modeling and filtration of models by SAXS and RDCs  PKI three-dimensional (3D) models consistent with the NMR-determined secondary structure were created using the MC-Fold/MC-Sym pipeline (Parisien and Major, 2008). The small-angle X-ray scattering amplitudes of the 629 models were predicted by using the FoXS web server (Schneidman-Duhovny et al., 2010). Agreement with the experimental SAXS amplitudes was evaluated by χ2 goodness-of-fit analysis. The top 25% of models, those with the lowest χ2 values, were then sorted by fit to 16 experimental imino 1H-15N RDC measurements as determined using the PALES/DC software (Zweckstetter and Bax, 2000). The models with a Q factor of <0.31 were chosen for future refinement. Covalent connectivity was restored to these five models using the AMBER force field in GROMACS (Open MM Zephyr software)(Van Der Spoel et al., 2005). The structures were simultaneously refined in XPLOR-NIH against SAXS and NMR data as previously described (Burke et al., 2012; Maori et al., 2007).    154  RESULTS Global structure of the IAPV IGR IRES PKI domain by SAXS analysis The overall fold of the PKI RNA was analyzed by small-angle X-ray scattering (SAXS). In order to delineate the PKI base pairing in the SAXS analysis, we compared the wild-type PKI RNA to a truncated RNA missing nucleotides 6604-6618 that cannot form a pseudoknot (Figure B.1 A-B, Table B.1).  The p(r) plot shows a major peak at 20 Å indicative of A-form RNA helical width, and indicates that the PKI has a larger maximum dimension (Dmax) consistent with pseudoknot formation (Figure B.1 D). The maximum dimension and radius of gyration (Rg) of PKI are 90 Å and 26.4 Å, respectively (Table B.1). PKIΔ6604-6618 has a 15 Å reduction in Dmax (75 Å) and an Rg of 22.8 Å, both consistent with its expected reduction in size.  NMR spectroscopy of IAPV PKI   The PKI secondary structure was determined from two-dimensional (2D) 1H-1H NOESY and 1H-15N HMQC NMR spectra in 20 mM potassium phosphate (pH 6.3), 200 mM KCl, and 0.5 μM EDTA  (Figure B.1 F and H). Aside from the expected loss of signals for nucleotides 6604-6618, deletion of PKI nucleotides 6604-6618 did not significantly alter the 1H-1H NOESY and 1H-15N HMQC spectra (Figure B.1 E and G), indicating that helices P3.1, P3.2, and P3.3 are folded in a similar manner in PKI and PKIΔ6604-6618. Nearly all base-paired imino resonances in PKIΔ6604-6618 (Figure B.1 E) and PKI (Figure B.1 F) were assigned, excluding those at helical termini that are rapidly exchanging with solvent. Sequential NOEs indicate formation of helix P3.1, P3.2, P3.3 and PKI within the PKI domain (Figure B.1 F).   In addition to all expected NOEs within helix P3.3 given the originally proposed base-pairing, an unexpected cross peak was detected between G6585 and U6586 (Figure B.1 E and F). These imino resonances are shifted upfield into a non-Watson-Crick region and suggest that the GUAACA is structured, most likely in a GNRA-type fold as this is a known motif that can tolerate insertions (consensus GNR(N)A;(Huppler et al., 2002; Zhang et al., 2016)). In helix P3.2, observation of the NOE cross-peak between G6568-U6570 indicates that U6569 is flipped out of the helix, allowing its 155  neighboring base-pairs to stack (Figure B.1 F). This conformation is consistent with reactivity levels obtained by SHAPE chemical probing for U6569 in the context of the PKI structure (Figure 3.6) (Ren et al., 2014). An additional resonance, tentatively assigned to U6562, was observable in the 1H NMR spectrum (Figure B.1 F) and in the 1H-15N HMQC (Figure B.1 H); however this resonance was not visible in the NOESY spectrum indicating that it exchanges with water during the 100 msec mixing time. The chemical shift is diagnostic of a U-G wobble pair (Barton et al., 2013; Chang et al., 1986), and the observed exchange broadening during the NOESY mixing time is consistent with tentative assignment of this imino resonance to U6562 which may form a U-G wobble pair with the terminal G6618. SHAPE probing on the PKI RNA showed moderate reactivity for U6562 and G6618 (see Figure 3.6 and (Ren et al., 2014)) consistent with transient base-pair formation. The stability of this base-pair in solution may affect the frequency of translation initiation in the +1 reading frame.   156    Figure B.1. Small-angle X-ray scattering and NMR analyses of IAPV IGR IRES PKI domain. Secondary structures of the wild-type IAPV IRES PKI domain (A) and PKIΔ6604-6618 (B). (C) Kratky profile and (D) pair distance distribution function plot of the wild-type and Δ6604-6618 IAPV IRES PKI domains. 1D 1H spectrum and 2D 1H-1H NOESY of the Δ6604-6618 (E) and wild-type (F) IAPV IRES PKI domains in 20 mM potassium phosphate (pH 6.3), 200 mM KCl, and 0.5 μM EDTA. 1H and 15N imino chemical shift assignments for Δ6604-6618 (G) and wild-type (H) IAPV IRES PKI domains. Assignments and connecting lines are color-coded according to secondary structure, as in (A) and (B). Base-pairs confirmed by 1H–1H 2D NOESY are indicated in (A) and (B) by black lines or circles, while base-pairs inferred by chemical shift agreement are indicated with gray lines.   157  Table B.1. Summary of SAXS data.  Sample PK (0.5-2.6 mg·mL-1) PK truncated (0.5-2.1 mg·mL-1) Data-collection parameters   Instrument APS – Sector 12-ID-B APS – Sector 12-ID-B Beam geometry Synchrotron Synchrotron Wavelength (Å) 0.866 Å 0.866 Å q range (Å-1) 0.005 0.760 Exposure time (s) 10 10 Concentration range (mgml-1) 0.5 – 2.6 0.5-2.1 Temperature (K) 298 298 Data-collection parameters   I(0) [from P(r)] 3.52 ± 0.02 X 10-2 5.27 ± 0.03 X 10-2 Rg (Å)[from P(r)] 26.5 ± 0.2 22.8 ± 0.2 I(0) (from Guinier) 3.5 ± 0.1 X 10-2 5.2 ± 0.1 X 10-2 Rg (Å) (from Guinier) 25.6 ± 0.3 22.3 ± 0.3 Dmax (Å) 90 ± 5 75 ± 5 Porod volume estimate 103 (Å3) 37.4 26.5 Dry volume calculated from sequence 103 (Å3) 21.4 16.8 Molecular-mass determination   Molecular mass [Vc] (kDa) 29.1 ± 4  24.3 ± 4 Calculated molecular mass from sequence (kDa) 22.6 17.8 Software Employed   Primary data reduction IGOR PRO IGOR PRO Data processing PRIMUS/GNOM PRIMUS/GNOM Ab initio analysis DAMMIF DAMMIF Validation and averaging DAMAVER DAMAVER Rigid-body modeling Mc-SYM Mc-SYM Computation of model intensities CRYSOL/FoXS CRYSOL/FoXS Three-dimensional graphics representations PyMol/Supcomb PyMol    158  APPENDIX C     Figure C.1. Ribosome binding assay for wild-type and SLVI mutant IRESs. Competitor RNAs (monocistronic wild-type, M1, M2 or M1+2 IAPV IRES) were added to 5'-end labeled wild-type IRES/80S ribosome complexes. The ability of competitor RNAs to compete for ribosome binding was determined by filter binding.  

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