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Intracellular inhibition of immune dysfunction induced by HIV-I NEF protein Chang, Alex Hongsheng 2000

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I N T R A C E L L U L A R I N H I B I T I O N OF I M M U N E D Y S F U N C T I O N I N D U C E D B Y HIV-1 N E F P R O T E I N  by ALEX HONGSHENG C H A N G M.D. The Capital University of Medical Sciences, China, 1986 M.Sc. The University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY A N D LABORATORY MEDICINE  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 2000 ©Alex Hongsheng Chang, 2000  Tuesday, August 8, 2000  UBC Special Collections - Thesis Authorisation Form  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  http://www.library.ubc.ca/spcoll/thesauth.html  Page: 1  ABSTRACT Current 'cocktail-therapy' toward HIV-1 infection using reverse transcriptase and protease inhibitors have been successful i n controlling the viral growth, but not very effective i n eradicating the reservoir of HIV-1 infected cells. It is a new challenge for H I V therapy to find ways to remove the virus reservoir that is composed of latently infected CD4+ T cells carrying integrated provirus. A potential new therapeutic target is Nef, a HIV-1 viral protein that downregulates class I M H C and by doing so it enables infected cells to elude killing by cytotoxic T lymphocytes.  In this thesis research, intracellular inhibition of Nef-mediated downregulation of C D 4 and M H C - 1 molecules was studied using recombinant single-chain antibodies (ScFvs) and a dominant-negative Hck. Several anti-Nef single-chain antibodies were first constructed. A l l retained the binding activity of their corresponding parental monoclonal antibodies when expressed intracellularly. However, ScFv expression was unable to inhibit C D 4 or M H C - 1 downregulation induced by Nef. This indicated that the intracellular binding of ScFv w i t h Nef and the following Nef sequestration may not be sufficient to prevent the receptor downregulation events induced by Nef. The expression of molecules capable of binding to epitopes i n Nef, that are implicated specifically i n receptor modulation, may be required for these effects.  ii  A dominant-negative form of H c k protein-tyrosine kinase, D N - H c k , composed of the H c k amino terminal region and its S H 3 and SH2 domains, was then studied as a potential candidate for preventing M H C - 1 downregulation; it is k n o w n that the H c k SH3 domain binds Nef w i t h a very high affinity (Kd=0.25 uM). In addition, the SH3-binding motif i n Nef, PXXP78 is also a major determinant i n downregulation of M H C - 1 . It was demonstrated that D N - H c k was able to block Nef-induced downregulation of class I M H C surface expression i n human cells. This effect required a functional SH3 domain, as it was not evident i n cells that expressed DN-Hck-W93F, an SH3 domain mutation that results i n diminished binding affinity for Nef. The results i n this thesis research thus support a model that D N - H c k prevents Nef-induced class I downregulation by blocking the interaction between Nef and an as yet unidentified SH3containing cellular protein that is capable of coupling Nef to the M H C - 1 molecule. U p o n binding with Nef, this cellular protein might recruit class I M H C molecules v i a a specific interaction w i t h their cytoplasmic motifs, which i n turn routes these molecules towards an intracellular degradation pathway. The SH3binding region of N e f therefore represents a new target for therapeutic intervention i n individuals infected with HIV-1.  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iv  TABLE OF FIGURES  vi  TABLE OF TABLES LIST OF ABBREVIATIONS  vii viii  ACKNOWLEDGEMENTS DEDICATION  xi xii  CHAPTER 1  1  INTRODUCTION  1  1.1 Foreword 1.2 Discovery of the AIDS virus 1.3 The immunopathogenesis of H I V infection 1.4 The challenge of H I V therapy 1.5 A n overview of H u m a n immunodeficiency virus type-1 1.6 A n overview of HIV-1 Nef protein 1.7 Structure of Nef protein 1.8 Nef downregulation of CD4 and its role i n H l Y - l infection 1.9 Nef downregulation of M H C - 1 and its role i n H I V evasion of the cellular immune response 1.10 The application of intracellular single-chain antibody i n H I V research 1.11 The interaction of H c k SH3 domain w i t h H I V - l Nef protein 1.12 Thesis objectives and hypotheses  1 4 7 11 16 27 29 33 38 42 44 48  CHAPTER 2  54  MATERIALS A N D METHODS  54  2.1 Materials 2.2 Inhibition ELISA assay 2.3 R N A isolation and reverse transcription-polymerase chain reaction 2.4 Cloning and sequencing of amplified products 2.5 Construction and expression of ScFv 2.6 Fluorescent microscopy 2.7 Characterization of expressed ScFv by immunoprecipitation 2.8 Co-transfection of Nef and ScFv or D N - H c k 2.9 F l o w cytometry analysis of C D 4 and M H C - 1 expression 2.10 S D S - P A G E and Western blot analysis of expressed protein  54 55 56 57 60 61 61 62 62 ..63  2.11 Immunoprecipitation of D N - H c k by immobilized Nef-GST fusion protein 63 CHAPTER 3  65  INTRACELLULAR SINGLE-CHAIN ANTIBODIES T A R G E T I N G NEF H A V E N O EFFECT O N NEF-INDUCED CD4 A N D MHC-1 D O W N R E G U L A T I O N .65 3.1 Introduction 65 3.2 Results 66 3.2.1 Monoclonal antibodies AG11 and AE6 bind to an overlapping epitope with EH1 66 3.2.2 Cloning and sequencing of mouse IgG variable regions 67 3.2.3 Construction and expression of intracellular ScFv tagged with a GFP reporter 72 3.2.4 CD4 and MHC-1 downregulation induced by Nef expression in P4.2 cells...78 3.2.5 Intracellular expression of ScFv EH1 and F14.ll did not affect receptor modulation induced by Nef. 78 3.3 Discussion '. 84 CHAPTER 4  91  H C K SH3 D O M A I N - D E P E N D E N T A B R O G A T I O N OF NEF-INDUCED CLASS I M H C D O W N R E G U L A T I O N  91  4.1 Introduction 91 4.2 Results 94 4.2.1 Dominant-negative Hck binds Nef through its SH3 domain 94 4.2.2 Differential rate of CD4 and MHC-1 downregulation induced by Nef. 99 4.2.3 DN-Hck inhibits class I MHC downregulation induced by Nef. 99 4.2.4 DN-Hck partially affects CD4 receptor downregulation by Nef 100 4.2.5 DN-Hck does not modulate CD4 or MHC-1 receptor expression by itself....104 4.2.6 Dose response of dominant-negative Hck in Nef expressing cells 105 4.3 Discussion 105 CHAPTER 5  112  DISCUSSION  112  5.1 Summary of results 5.2 Discussion and conclusion BIBLIOGRAPHY  112 113 127  v  T A B L E OF FIGURES Figure 1 Typical clinical course of H I V infection 8 Figure 2 Current H I V therapy 15 Figure 3 Organization of the HIV-1 genome and virion 17 Figure 4 HIV-1 replication cycle 19 Figure 5 HIV-1 reverse transcription 22 Figure 6 HIV-1 R N A splicing 25 Figure 7 Nef-SH3 interaction 30 Figure 8 Sequence alignment of Nef core 31 Figure 9 Sequence alignment of SH3 domains 32 Figure 10 The mechanism of C D 4 downregulation induced by Nef 36 Figure 11 Domains of Nef implicated i n receptor downregulation 37 Figure 12 Structure of Src family tyrosine kinases 45 Figure 12a Single-chain antibody construction 58 Figure 13 Binding inhibition of biotinylated E H 1 M a b by unlabeled A G 1 1 and A E 6 Mabs 68 Figure 14 Alignment of the c D N A sequences of variable regions derived from clones A G 1 1 , A E 6 and E H 1 69 Figure 15 Deduced amino acid sequence alignment of the variable regions of clones A G 1 1 , A E 6 and EH1 71 Figure 16 The single chain antibody assembly i n p D E F - G F P expression vector. .74 Figure 17 H E K 293 cells transfected with p D E F - G F P or pDEF-ScFv-GFP 75 Figure 18 Intracellularly expressed ScFv is immunoprecipitated by immobilized recombinant Nef-GST protein 77 Figure 19 C D 4 and M H C - 1 modulation i n response to Nef 79 Figure 20 The single chain antibody assembly i n pDEF-myc expression vector. .81 Figure 21 Co-expression of single chain antibodies and Nef i n P4.2 cells 83 Figure 22 F l o w cytometry analysis of C D 4 and M H C - 1 receptor expression i n P4.2 cells co-transfected with Nef and ScFv constructs 85 Figure 23 The dominant-negative Hck, and its SH3 and SH2 mutant-constructs (Tokunaga et al, 1998) 95 Figure 24 A functional H c k SH3 domain is required for efficient binding of Nef to DN-Hck 96 Figure 25 Co-expression of D N - H c k mutant proteins and Nef i n P4.2 cells 98 Figure 26 The dominant-negative H c k blocks the C D 4 and M H C - 1 cell surface molecule downregulation effect induced by Nef 101 Figure 27 Summary of C D 4 and M H C - 1 receptors downregulation-blocking effect by dominant negative H c k 103 Figure 28 The dominant-negative H c k alone does not modulate C D 4 and M H C - 1 cell surface molecule expression 106 Figure 29 The dominant-negative H c k blocks the M H C - 1 cell surface molecule downregulation induced by Nef i n a dose-dependent manner 108 Figure 30 M o d e l for the dominant-negative H c k inhibition of M H C - 1 receptor downregulation induced by Nef 119 ;  vi  T A B L E OF TABLES Table 1 Comparison of H I V and H T L V : Table 2 Comparison of the variable region sequences of clones A E 6 and E H 1 withAGH  72  LIST OF A B B R E V I A T I O N S AIDS  -acquired immunodeficiency syndrome  AP-1  adaptor protein complex-1  AP-2  adaptor protein complex-2  P-COP-I  (3 subunit of coat protein-I  CA  capsid protein (p24 i n HIV)  CD4  clusters of differentiation-4  CDR  complementarity determining region  CTL  cytotoxic T-lymphocyte  DN-Hck  dominant-negative H c k  E F - l a promoter  Elongation factor-la promoter  ELISA  enzyme-labeled immunosorbant assay  ER  endoplasmic reticulum  Env  proteins encoded by envelope orf  Gag  protein encoded by group-specific antigen open  reading frame GFP  green fluorescent protein  GST  glutathione S-transferase  HAART  highly active anti-retroviral therapy  Hck  hematopoietic cell kinase  HLA  human leukocyte antigen  HIV-1 HIV-2 HTLV  :  human immunodeficiency virus-1 human immunodeficiency virus-2 -.—human T-cell leukemia virus  IN  integrase  LAV  lymphoadenopathy-associated virus  LTNP  long term non-progressor  LTR  long terminal repeat  MA  matrix protein (pl7 i n HIV)  Mab  monoclonal antibody  MHC  major histocompatibility complex  NC  nuclear capsid protein (p7 in HTV)  NK  natural killer cells  Nef  negative factor  NMR  nuclear magnetic resonance  orf  open reading frame  PBMC  peripheral blood mononuclear cells  PBS  :  phosphate buffered saline  PCR  polymerase chain reaction  PHA  phytohemagglutinin  Pol  protein encoded by polymerase open reading frame  PR  protease  Rev  regulator of viral expression  RRE  Rev responsive element  RT  reverse transcriptase  RT loop  a region of the SH3 domain which has functionally  important arginine and threonine residues. ScFv  single-chain (variable fragment) antibody.  SCID  severe combined immunodeficiency. ix  SH2  Src homology domain 2  SH3  Src homology domain 3  SrV  simian immunodeficiency virus  Src  src oncogene of Rous sarcoma virus  SU  surface protein (gpl20 i n HIV)  TAR  frans-activating response element  Tat  fnms-activator of viral trahscriptiion  TCR  T cell receptor  TGN  rrans-Golgi network  nyi  transmembrane protein (gp41 i n H I V )  Vif  viral infectivity factor  V  variable region of the heavy chain  H  VK  variable region of the kappa light chain  Vpr  viral protein R  Vpu  viral protein U  x  ACKNOWLEDGEMENTS I w o u l d like to thank the following people who have either contributed to this thesis research or supported and helped during this long journey. I w o u l d like to thank Dr. Sharon Cassol (relocated to Ottawa General Hospital i n 1996), m y former supervisor who first pointed out to me that Nef was a very interesting H I V viral protein to study. Thanks to Dr. Julian Davies, who I had worked w i t h as a research assistant for one year i n 1994, and to w h o m I have great respect as a scientist, and w h o has encouraged and supported my quest throughout this graduate study. I am very grateful to Drs. K e v i n Leslie, James Hoxie, Mark Harris, Rita De Santis, Oliver Schwartz and M i c h i y u k i Matsuda for generously providing reagents that were essential for this study. Special thanks to Dr. James Hoxie w h o was k i n d enough to communicate w i t h me numerous times through email to discuss research problems and exchange ideas during the first part of this thesis research (single-chain antibody construction and evaluation). Special thanks to Dr. Chris O n g for the insightful discussions we had i n the lab. Thanks also to several key people i n Frank's lab, Jim Peacock, Lettie Hsiao, Elizabeth Hajen, Lorraine Spence, for their instrumental, technical and lab support. M a n y thanks to people i n this department, Dr. Donald Brooks, Penny W o o , Charles Ramey, Vanessa Lowe, for their support and assistance during this graduate studies. I am very grateful to m y graduate committee, Drs. Susan Porter, Jaki Chantler, Julio Montaner, Keith Humphries, Julian Davies, Michael O'Shaughnessy, and Frank Jirik, for their support and encouragement. Special thanks to Keith Humphries, w h o was very patient and helpful i n advising me during the writing of this thesis. Last, but not least, I w o u l d like to thank my thesis supervisors, my research supervisor, D r . Frank Jirik, for picking me up as a graduate student after Sharon left t o w n i n 1996, for his tolerance to allow me to continue m y project, and for his guidance i n this thesis research. Thanks to Dr. Michael O'Shaughnessy, m y co-supervisor, w h o encouraged and supported me throughout this program. This w o r k was supported i n part by the British Columbia Center of Excellence i n H I V / A I D S , and by the National Health Research and Development Program through a National Health Fellowship to A . H . C .  DEDICATION  To my parents, who are not here to see this day, I hope that I can make y o u proud.  And  To m y wife, Yanbo, Thanks for your patience, I promise that we w i l l have a normal life from n o w on.  And  To my two boys, Arthur and Eddie, Y o u make me feel young and energized everyday.  xii  CHAPTER 1 INTRODUCTION 1.1 Foreword Nef is an important regulatory protein of the primate lentiviruses, human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV). A l t h o u g h the exact role of Nef i n the life cycle of lentiviruses remains to be fully elucidated, it is known that Nef is essential for high viral load and increased disease progression i n rhesus monkey animal models infected w i t h SIV, as well as individuals infected w i t h HIV-1 (Deacon et al, 1995; Kirchhoff et al, 1995). Several distinct functions of Nef have been characterized in vitro. First, Nef downregulates the cell surface expression of C D 4 and M H C - 1 receptors (Piguet et al., 1999b). Second, Nef increases viral infectivity at a stage after entry of the virus into the cell (Aiken and Trono, 1995; Chowers et al., 1995; Schwartz et al., 1995). Nef also alters cellular signal transduction and activation pathways (Baur et al, 1994; Hanna et al, 1998; Iafrate et al, 1997; Skowronski et al, 1993).  Nef, i n concert w i t h E n v and V p u , downregulates C D 4 receptors. This function may serve to inhibit superinfection and increase the infectivity of the viruses (Piguet et al, 1999b). Nef-induced M H C - 1 downregulation may play an important role i n the pathogenesis of HIV-1 infection through immune escape by protecting infected cells from recognition of cytotoxic T lymphocytes (Collins et al, 1998).  1  The mechanisms used by Nef i n CD4 and M H C - 1 downregulation are different. Moreover, Nef uses distinctive determinants for the two activities. Nef downregulates C D 4 by acting as a connector between the receptor and intracellular trafficking pathways causing CD4 internalization (Mangasarian et al, 1997). Nef associates w i t h the CD4 cytoplasmic tail by recognizing the dileucinebased motif that also functions as an endocytosis signal (Aiken et al., 1994; H u a and Cullen, 1997). The immediate downstream partner of Nef for C D 4 downregulation is the clathrin-associated adaptor protein complex-2 (AP-2) (Piguet et al., 1999b). The internalized C D 4 molecules are then targeted for lysosomal degradation through an interaction w i t h (3 subunit of coat protein-I (p-COP-I) i n endosomes (Piguet et al, 1999a).  The pathway through which M H C - 1 molecules are modulated by Nef is yet to be elucidated. N o direct association has been found between Nef and M H C - 1 (Le Gall et ah, 1998). A cryptic tyrosine-based sorting signal motif i n the cytoplasmic domain of H L A - A and -B heavy chains was revealed, i n connection w i t h M H C - 1 downregulation i n the presence of Nef (Le Gall et ah, 1998). The H L A - C molecules, w h i c h do not bear the tyrosine-sorting signal motif, are not affected by Nef (Le G a l l et al, 1998). The adaptor protein complex-1 (AP-1) may be involved i n the M H C - 1 downregulation by Nef, since AP-1 co-localizes w i t h M H C - 1 i n the rrans-Golgi network (Greenberg et al, 1998b; Le Gall et al, 1998) and binds w i t h Nef i n a yeast two-hybrid system as well as i n cell-free assays (Le Gall et al, 1998).  2  The ability of Nef to downregulate M H C - 1 receptor expression requires a wellconserved Src homology 3 (SH3) domain-binding P X X P motif i n the Nef core (Greenberg et al, 1998b), w h i c h is dispensable for the down-modulation of C D 4 receptors by Nef (Goldsmith et al, 1995; Saksela et ah, 1995). This indicates that a cellular protein w i t h an SH3 domain capable of interaction w i t h Nef may be involved i n the M H C - 1 modulation activity. Several tyrosine kinases of the Src family b i n d to the polyproline (PXXP78) motif i n N e f via their SH3 domains (Lee et al, 1996; Saksela et al, 1995). Full-length Nef binds to the H c k SH3 domain w i t h the highest affinity reported for an SH3-mediated interaction ( K d = 0.25 uM) (Lee etal, 1995). '  The research described in this thesis uses two approaches to inhibit the effects of Nef expression i n the host cells as indicated by C D 4 and M H C - 1 expression. One approach is to use recombinant intracellular single-chain antibody (ScFv), w h i c h has been successfully employed to study the function of cytosolic proteins by either blocking the function or sequestrating the protein of interest. ScFvs, w h i c h have been shown to have specific binding affinities equivalent to those of the parent monoclonal antibodies (Whitlow, 1991b; Winter and Milstein, 1991), can be stably expressed intracellularly where they are capable of inactivating specific cellular gene products (Biocca et al, 1990; Carlson, 1988). Intracellular ScFv proteins w i t h specificity for virally-encoded proteins thus provide a unique way of studying the role of these viral proteins i n HIV-1 infection (Marasco et al, 1993), as w e l l as offering a potential gene therapy strategy for inhibiting the development of AIDS.  3  Another approach is to use a dominant-negative form of Hck, a protein tyrosine kinase that was shown to have high binding affinity with Nef through its SH3 domain. Dominant-negative H c k (DN-Hck) consisting of the H c k aminoterminal domain, together w i t h its SH3 and SH2 domains has been co-expressed w i t h proviral D N A i n HIV-1 producer cells and the virus so produced has been shown to have reduced viral infectivity (Tokunaga et al., 1998). In addition to the proposed mechanism that D N - H c k may bind to the Src family kinases and hence inhibit their enzymatic activity (Tokunaga et al, 1998), it may also reduce viral infectivity through binding w i t h the polyproline motif i n Nef and thereby blocking the downstream interaction of cellular proteins w i t h Nef. The latter mechanism raises the possibility that the SH3-binding motif i n Nef may be a good therapeutic target, since this motif is involved i n class I molecule downregulation and several other detrimental effects of Nef on the host cells i n HIV-1 infection (Arold et al, 1997; Briggs et al, 1997; Collette et al, 1996; Goldsmith et al, 1995; Greenberg et al, 1998b; Mangasarian et al, 1999).  1.2 Discovery of the AIDS virus A I D S (acquired immunodeficiency syndrome) was first found to be caused by a retrovirus i n 1983, when scientists at the Pasteur Institute recovered a reversetranscriptase-containing virus from the l y m p h node of a patient w i t h persistent lymphadenopathy; accordingly, this virus was designated lymphadenopathyassociated virus (LAV) (Barre-Sinoussi et al, 1983; Wain-Hobson et al, 1991). Lymphadenopathy-associated virus was shown to replicate and cause cytopathology i n cultures of human peripheral blood lymphocytes. A t the same time, human T-cell leukemia virus (HTLV) was reportedly isolated from  4  individuals w i t h AIDS and was suggested to be the causative agent (Gallo et al, 1983). Further studies i n 1983 by Montagnier and coworkers refuted the notion that H T L V is the virus responsible for A I D S (Chermann et ah, 1983). Their results indicated that this retrovirus, although similar to H T L V i n infecting CD4+ lymphocytes, had quite distinct properties. The virus they isolated grew to substantial titer i n CD4+ cells and killed them instead of transforming the cells i n culture as H T L V does. In early 1984, Gallo and associates reported another H T L V that has the characteristics of a human retrovirus with lymphotropic and cytopathic properties. Levy and coworkers also reported, i n 1984, the identification of retroviruses recovered from A I D S patients from different k n o w n risk groups (Levy et al., 1984). These viruses were later confirmed to be members of the same group of retroviruses from lentivirinae that had many properties distinguishing them from H T L V (Table 1) (Levy, 1994). In 1986 the International Committee on Taxonomy of Viruses recommended giving the A I D S virus a separate name, the human immunodeficiency virus (Levy, 1994).  Table 1 Comparison of HIV and H T L V (Table reproduced from Levy, 1994) Characteristic  HIV  HTLV  Retrovirus genus  Lentivirus  HTLV/BLV  Genome size (kb)  9.8  9.0  Core morphology  Cone  Cuboid  Accessory genes  6  2  Cytotoxic  +  Cell transformation  +  5  H I V isolates were subsequently recovered from the blood of many patients w i t h AIDS, as well as from the P B M C of clinically healthy individuals (Levy et. al., 1985; Salahuddin et al., 1985). Soon after the discovery of HIV-1, a separate virus type, HIV-2, was identified i n western Africa (Clavel et ah, 1986). Although both of these viruses cause AIDS, individuals infected with HIV-2 exhibit a longer period of clinical latency and lower morbidity (Essex M , 1994).  The discovery of additional distinct lentiviruses i n non-human primates as well as in humans has provided important insights into the biologic significance and evolutionary relationships of these viruses. In 1985, a lentivirus was isolated from captive Asian Macaques with an AIDS-like disease (Daniel et al., 1985) and was later designated as simian immunodeficiency virus (SIV) because of its morphologic similarity and serologic cross-reactivity. Additional lentiviruses have since been isolated from several monkey species i n the w i l d throughout several regions i n Africa (Hirsch V M , 1993) i n the indigenous or natural host, these viruses do not produce disease (Gardner M B , 1994). SIV is the closest k n o w n animal-virus relative of H I V , although it is only about 50% related on the basis of sequence analysis. Interestingly, several HIV-2 isolates of West African origin are almost indistinguishable at the nucleotide sequence level from certain strains of SIV (Gao et al., 1992), indicating that the primate and human viruses share evolutionary roots and that there might have been interspecies infection (Essex and Kanki, 1988).  6  1.3 The immunopathogenesis of HIV infection  The typical course of H I V infection is characterized by multiple phases that occur over a period of eight to ten year (Abrams et al, 1984; Buchbinder et al., 1994; Fauci, 1991; Lifson et al., 1991). It generally includes three phases 1) primary infection, which is characterized in approximately 50-70% of infected individuals by nonspecific symptoms such as fever, lethargy, sore throat, myalgias, lymphadenopathy, and macupapular rash (Clark et al., 1991; Daar et al., 1991; Tindall and Cooper, 1991); 2) clinical latency, which varies i n length, with an average of 10 years, during which there is lack of clinical symptoms; 3) A I D S disease phase, which is characterized by low peripheral blood CD4+ T cell counts (<200 per (ii), severe and persistent constitutional symptoms and increased susceptibility to neoplasms and opportunistic infections.  Based on the clinical course of H I V infection, several subgroups of patients have been identified (Borrow et al, 1994; Collins and Baltimore, 1999; K o u p et al, 1994; Pantaleo and Fauci, 1996). These include typical progressors w h o represent the majority of HIV-infected individuals (Figure 1). The median time from initial infection to progression to A I D S i n typical progressors is eight to ten years (Buchbinder et al, 1994; Fauci, 1991; Lifson et al, 1991). Subjects w h o have an unusually rapid progression of disease are called rapid progressors. Those w h o do not experience progressive disease for several years (eight to ten) following primary infection are called long-term nonprogressors. Finally, those w h o progress to A I D S within a time frame similar to typical progressors but w i t h stable clinical and laboratory parameters for unusually long period of time once disease progression has occurred are named long-term survivors.  7  Weeks  Years  Figure 1 Typical clinical c o u r s e of HIV infection on the b a s i s of the c h a n g e s of C D 4 + T-cell c o u n t s a n d v i r e m i a over time. Following primary infection, typical p r o g r e s s o r s e x p e r i e n c e a long period of clinical latency. P r o g r e s s i o n to A I D S generally o c c u r s within eight to ten y e a r s . • C D 4 T cell count, • Culturable p l a s m a viremia, • HIV R N A C o p i e s per ml p l a s m a . (Figure r e p r o d u c e d from P a n t a l e o e r a / . , 1994).  H I V infection occurs at the sites of bloodstream or mucosa. Following the initial peak of viremia, HIV-specific humoral and cell-mediated immune responses are readily detected (Borrow et al, 1994; Graziosi et al, 1993; K o u p et al, 1994; Moore et al, 1994; Pantaleo et al, 1994a; Reimann et al, 1994). A variety of anti-HIV antibodies and cytotoxic T lymphocytes (CTLs) specific for different H I V proteins •i  are detected very early during primary infection. H i g h titers of antibodies specific for a variety of H I V proteins are a major component of the primary immune response to the virus (Clark et al, 1991; Daar et al, 1991; Moore et al, 1994; Tindall and Cooper, 1991). However, the H I V antibodies produced during primary infection lack neutralizing activity against HIV-1 infection (Koup et al, 1994; Moore et al, 1994). Therefore, these antibodies may have little effect on initial virus dissemination and the primary HIV-specific antibody response is generally considered nonprotective. However, this antibody response may contribute significantly to the dramatic reduction of viremia by trapping the circulating virus particles in the l y m p h tissue. This notion was supported by the SIV animal model of acute infection, w h i c h demonstrated that the decline of H I V viremia coincides w i t h a rise i n HIV-specific antibody titers, and w i t h the intensity of virus trapping in l y m p h nodes (Pantaleo and Fauci, 1995; Pantaleo et al, 1994b).  HIV-specific C T L s are considered to play a major role i n the initial suppression of virus replication. Virus-specific cytotoxic CD8+ T cells can be detected as early as five days following infection, as indicated by experiments performed i n the SIV model of acute infection (Reimann et al, 1994). Similarly, i n most subjects w i t h primary H I V infection, virus-specific CTLs are consistently and readily detected  9  in P B M C during the acute viral infection, and their appearance correlates w i t h the reduction of viremia. In general, HIV-specific cytotoxic activity may be detected against both structural (Env and Gag) and regulatory (Tat and Rev) proteins of H I V . However, during the primary infection H I V specific cytotoxic activity is detected predominantly against structural proteins. In addition to mediating cytotoxic activity, it is also demonstrated that CD8+ T cells from subjects w i t h primary H I V infection release a number of soluble factors (chemokines, such as R A N T E S , M l P - l a , M I P - i p and IL-16) w i t h suppressing activity on virus replication (Baier et al., 1995; Cocchi et al., 1995). Therefore, CD8+ T lymphocytes may contribute to the suppression of viremia either by elimination of virus-infected cells via a cytotoxic mechanism or by soluble factormediated suppression of virus replication.  Despite the early appearance of a vigorous immune response, this immune reaction ultimately fails to eliminate the virus since transition to the chronic phase of infection occurs i n most individuals (Pantaleo et al., 1993b). Furthermore, although a dramatic suppression of viremia is observed i n infected individuals as progression to the chronic phase of infection occurs, virus replication is never completely curtailed. It has been well established i n recent studies that H I V actively and continuously replicates even during the period of clinical latency, and that lymphoid organs serve as the primary site of virus replication (Embretson et al., 1993; Pantaleo et al., 1991; Pantaleo et al., 1993a; Piatak et al., 1993). These observations indicated that H I V disease is active and progressive throughout the course of infection and that clinical latency is not equivalent to silence in disease. This persistent virus replication and the chronic  10  immune activation associated w i t h it have been proposed to be responsible for the progressive destruction of lymphoid tissue, and hence the deterioration of the immune system (Fauci, 1993; Pantaleo and Fauci, 1994).  In favor of this hypothesis, recent studies have demonstrated that virus replication and turnover are extremely high, and that there is a direct correlation between the level of viremia and changes in CD4+ T cell counts (Ho et al., 1995; W e i et al, 1995). It has been suggested that there is an extremely high turnover of CD4+ T cells and that a large number of CD4+ T cells are depleted and replaced daily. However, the mechanisms responsible for this rapid increase of CD4+ T cells following virus suppression remain to be elucidated.  Taken together, this knowledge and observations have greatly contributed to a better understanding and delineation of the pathogenic mechanisms leading to progression of H I V disease, and they have provided great insights for the current 'cocktail' antiviral therapy and for the future development of therapeutic strategies.  1.4 The challenge of HIV therapy  Despite the decline of deaths from AIDS i n industrial countries, that stemmed mainly from the introduction of powerful therapies able to retard the activity of H I V i n 1996, the international pandemic of H I V infection and A I D S is still expanding rapidly i n the developing nations where the vast majority of people reside (Mann and Tarantola, 1998). According to new estimates from the joint United Nations Program on H I V / A I D S (UNAIDS) and the W o r l d Health  11  Organization (WHO) (AIDS epidemic update: December 1999, U N A I D S Joint United Nations Program on H I V / A I D S ) , 32.4 million adults and 1.2 million children were living w i t h H I V by the end of 1999, and almost 16.3 million people have died since the beginning of the epidemic i n early 1980s. In 1999 alone, some 5.6 million people, close to 15,000 people a day, acquired H I V , and some 2.6 million perished from it, including 470,000 children. Control of the pandemic w i l l require extensive prevention programs and more effective and economic therapies.  H I V infection presented a new challenge to modern medicine: the retroviruses can integrate into the genome of host cells, where they can lie dormant for as long as the life of the host cells. Without effective therapeutic intervention, H I V infection is almost invariably a progressive, lethal disease that completely destroys the patient's immune system and causes death of the infected individual from opportunistic infections.  Better understanding of how H I V behaves in the body and more choice of drugs i n the anti-HIV arsenal have led to several break-throughs i n medical history that provide m u c h brighter prospects for most patients who receive treatment today (Bartlett and Moore, 1998). W i t h the advance of highly sensitive viral detection technology, it is clear n o w that H I V replicates prolifically from the onset of the infection. H I V levels remain fairly stable for several years only because the body responds to the infection by manufacturing extraordinary numbers of CD4+ T cells. Patients who display cellular immunity usually have lower viral load and their disease course usually progress more slowly than  12  individuals w h o mount a weaker immune response. In addition, viral levels seem to correlate w i t h prognosis. Such findings indicate that the amount of virus or the viral load i n the host immune system plays a major role i n determining disease progress. Therapy must therefore aim to suppress viral replication immediately following infection and throughout the disease course.  So far, all approved anti-TflV drugs attempt to block viral replication within cells by inhibiting either reverse transcriptase or the H I V protease. There are two classes of drugs that inhibit reverse transcriptase and thus prevent viral R N A copying itself into c D N A for viral integration. The nucleoside analogues resemble the natural substances that become building blocks of H I V D N A and terminate the reverse transcription process when added to a developing strand of H I V D N A . This group includes the first anti-HIV drug-zidovudine (AZT) that was introduced i n 1987, and its close chemical relatives. Non-nucleoside reverse transcriptase inhibitors such as nevirapine bind close to the polymerase active site and thereby keep the RT inactive. A relatively new class of drugs, protease inhibitors, block the active catalytic site of the H I V protease and thereby preventing it from cleaving newly translated H I V proteins and hereby inhibit the subsequent viral assembly.  The current optimal therapy to achieve maximum viral suppression is the 'cocktail-therapy' or H A A R T (highly active antiretro viral therapy) (Figure 2). A t the moment, H A A R T usually consists of triple therapy, including two nucleoside analogues and a protease inhibitor (Bartlett and Moore, 1998). Treatment w i t h these potent antiretroviral regimens can produce sustained suppression of H I V -  13  1 replication, w i t h reduction of H I V R N A i n infected individuals to below the limits of detection i n blood for two or more years (Gulick et ah, 1997). Patients receiving H A A R T have seen their CD4 + T cells rebound significantly as a sign of the immune system i n recovery (Autran et al, 1997).  While the powerful combination therapies can suppress H I V i n the blood stream of infected patients to a level below the limits of detection using the most sensitive detection methods, the virus continues to persist i n a dormant form i n a small number of CD4+ T lymphocytes. This reservoir of latently infected cells does not die out and take the virus with them into eradication, as many A I D S researchers had predicted (Perelson et al., 1997). Clinical evidence has shown that H I V continues to replicate at a low level even i n patients w i t h undetectable levels of virus i n their blood (Ho, 1997). Moreover, the progeny viruses are wild-type rather than drug-resistant strains, indicating that even viruses sensitive to the drugs are not completely suppressed.  The key issue for virus eradication seems to be removing the virus reservoir that is composed of latently-infected memory CD4+ T cells carrying integrated provirus (Chun et al, 1997; C h u n et al, 1995; Coffin, 1995). Post-integration latency appears to result from the reversion of productively infected CD4+ T lymphoblast to a resting memory state i n which there is minimal transcription of viral genes. Because memory CD4+ T cells can persist for months to years (McLean and Michie, 1995; Michie et al., 1992), resting memory CD4+ T cells carrying replication-competent viral genomes may represent an important longterm viral reservoir i n patients on H A A R T . More research is required to  14  Figure 2 Current HIV therapy. Current HIV drugs aim to stop viral replication by inhibiting reverse transcriptase or p r o t e a s e . Other drugs under investigation a r e a l s o s h o w n . (Reprint with p e r m i s s i o n , from T o m o N a r a s h i m a , M e d i c a l & S c i e n c e Illustrator a n d Scientific American).  15  overcome this problem as current therapy is hot effective i n eradicating provirus integrated i n resting T lymphocytes.  In this thesis research, Nef is examined as a potential drug target, since one of its detrimental effects i n the infected cells is to downregulate M H C - 1 and i n doing so it enables virus infected cells to elude killing by cytotoxic T-lymphocytes. interfering w i t h Nef-induced class I M H C downregulation may therefore represent a novel strategy for increasing HIV-1-specific cytotoxic cell activity against infected cells. Therapies designed to inhibit M H C - 1 downregulationinduced by Nef w i l l be potentially beneficial to patients i n all stages of H I V infection, since its main function is to make the infected cells more 'visible' to the CTLs-cellular immune system that is responsible to clear virus infected cells from the immune system. It is hopeful that together w i t h other powerful anti-HIV treatment w h i c h inhibits viral replication, they may eventually reach the goal of curing H I V infection.  1.5 An overview of Human immunodeficiency virus type-1  H u m a n immunodeficiency virus type 1 (HIV-1) has been the subject of intense investigation for almost two decades, and a great deal has been learned about how the retrovirus infects cells, replicates, and causes disease (reviewed i n (Frankel and Young, 1998)).  The HIV-1 genome encodes nine open reading frames (Figure 3). Three of these encode the Gag, Pol, and Env polyproteins, w h i c h are subsequently proteolyzed into i n d i v i d u a l proteins common to all retroviruses. The four Gag proteins,  16  env LTR  Figure 3 O r g a n i z a t i o n of the HIV-1 g e n o m e and virion. T h e HIV-1 g e n o m e is a r o u n d 9 kb a n d e n c o d e s nine o p e n reading frames. T h r e e of t h e s e e n c o d e the G a g , P o l , a n d E n v polyproteins, w h i c h are subsequently proteolyzed into individual proteins c o m m o n to all retroviruses. T h e four G a g proteins, matrix ( M A ) , c a p s i d ( C A ) , n u c l e o c a p s i d ( N C ) , a n d p6, a n d the two E n v proteins, g p 1 2 0 or surface protein ( S U ) a n d gp41 or t r a n s m e m b r a n e protein (TM), are structural c o m p o n e n t s that m a k e up the c o r e of the virion a n d outer m e m b r a n e e n v e l o p e . . T h e HIV-1 g e n o m e is a r o u n d 9 kb a n d e n c o d e s nine o p e n reading f r a m e s . T h r e e of these e n c o d e the G a g , P o l , a n d E n v polyproteins, which are s u b s e q u e n t l y proteolyzed into individual proteins c o m m o n to all retroviruses. T h e four G a g proteins, matrix (MA), c a p s i d ( C A ) , n u c l e o c a p s i d ( N C ) , a n d p6, a n d the two E n v proteins, g p 1 2 0 or surface protein (SU) a n d gp41 or t r a n s m e m b r a n e protein (TM), are structural c o m p o n e n t s that m a k e up the c o r e of the virion a n d outer m e m b r a n e e n v e l o p e . T h e three P o l proteins, protease ( P R ) , r e v e r s e transcriptase (RT), a n d integrase (IN), provide essential enzymatic functions a n d are a l s o e n c a p s u l a t e d within the particle. HIV-1 a l s o e n c o d e s six regulatory proteins, three of w h i c h (Vif, V p r a n d Nef) are found in the viral particle. Two other regulatory proteins, Tat a n d R e v , provide e s s e n t i a l g e n e regulatory functions, and the last regulatory protein, V p u , a s s i s t s in a s s e m b l y of the virion by releasing E n v from e n d o p l a s m i c reticulum. (Reprint with p e r m i s s i o n , from the A n n u a l R e v i e w of Biochemistry V o l u m e 67, 1998 by Annual Reviews www.AnnualReviews.org).  17  matrix ( M A ) , capsid (CA), nucleocapsid (NC), and p6, and the two Env proteins, gpl20 or surface protein (SU) and gp41 or transmembrane protein (TM), are structural components that make up the core of the virion and outer membrane envelope. The three P o l proteins, protease (PR), reverse transcriptase (RT), and integrase (IN), provide essential enzymatic functions and are also encapsulated within the particle. HIV-1 also encodes six accessory or regulatory proteins, three of w h i c h (Vif, V p r and Nef) are found i n the viral particle. Two other accessory proteins, Tat and Rev, provide essential gene regulatory functions, and the last accessory protein, V p u , assists in assembly of the virion by releasing E n v from endoplasmic reticulum (ER). The retroviral genome is encoded by viral R N A of around 9 kb, and two identical viral genomic R N A molecules are packaged i n each virus.  The cycle of HIV-1 infection and replication is involved with the following distinct steps (reviewed i n (Frankel and Young, 1998)) (Figure 4). V i r a l entry (Step 1 and 2, Figure 4) is initiated by the binding of the S U glycoprotein, located on the viral membrane surface, to specific cell surface receptors. The major receptor for HIV-1 is C D 4 , an immunoglobulin-like protein expressed on the surface of a subset of T cells and primary macrophages . The SU-CD4 interaction alone is not sufficient for HIV-1 entry. Instead a group of chemokine receptors (a family of seven transmembrane G-coupled proteins, e.g. C X C R 4 , C C R 5 , C C R 3 , etc.) serve as essential viral coreceptors (Berger et al, 1999). Following binding, the transmembrane protein (TM), gp41, undergoes a conformational change that promotes virus-cell membrane fusion, thereby allowing entry of the viral core into the cell.  18  Figure 4 HIV-1 replication cycle. HIV-1 infection begins with virus binding to a susceptible target cell via a specific interaction between the viral gpl20 envelope glycoprotein and the C D 4 cellsurface receptor (step 1) (Young, 1997). Following binding, a process of membrane fusion, facilitated by the viral gp41 envelope glycoprotein and by the H I V coreceptors, C X C R 4 O R C C R 5 , results i n the introduction of the HIV-1 core particle into the cell cytoplasm (step 2). In activated and proliferating Tlymphocytes, reverse transcription of the viral R N A (step 3) and the integration of resulting D N A copy into the host-cell chromosome ensues (step 4). In resting cells, however, these events proceed inefficiently, if at all. Once integrated i n the chromosome, the transcriptional activity of the HIV-1 provirus is regulated by constitutive host-cell transcription factors (i.e., S p l and the T A T A - b i n d i n g factors), the activation-inducible members of the N F - k B family of host transcription factors (i.e., p50 and p65), as well as the virally encoded tat protein (step 5). Following synthesis of a full-length viral R N A , a complex array of alternatively spliced viral m R N A s can be produced. The differential expression of distinct species of viral m R N A s is controlled by the HIV-1 Rev protein. The level of Rev present i n an infected cell determines the preferential production of either the unspliced or singly spliced R N A s that provide viral R N A genomes or encode essential structural or enzymatic proteins (i.e., Gag, Pol, and Env), or the multiply spliced m R N A s that encode the viral regulatory gene products (i.e., Tat, Rev, and Nef). In circumstances i n which the amount of Rev present in an infected cell is limiting, such as the early stages of viral infection or when the overall level of transcription is low, only the multiply spliced m R N A transcripts are available i n the cytoplasm for the translation of viral proteins (step 6). Once a sufficient level of rev accumulates, the singly spliced and unspliced FTTV-l R N A s appear in the cytoplasm, and the synthesis of viral structural proteins can proceed (step 7). HIV-1 particles assemble at the host-cell surface (step 8), and they acquire viral env proteins as they bud through the host-cell membrane. The viral Gag and Gag-Pol polyproteins are cleaved by viral protease during or shortly after budding, generating mature infectious virions (step 9). (Reprinted w i t h permission, from Young, J.A.T. The Replication Cycle of HIV-1 In P.T. Cohen, M . A . S . , Paul A . Volberding (ed.) A textbook on HIV disease from the University  of California,  San Francisco and San Francisco General Hospital.  http://hivinsite.ucsf.edu/akb/1997/index.html).  19  ;CiV; HIV Ertv-CEM Binding _ t&i IZiMembrane Fusion CD4 fj|y„| and Entrv  ^ |Sf T* <  stranded RNA  3. Reverse Transcription  Mul  Q D — C M  «==*  v  ru5  Linear double U3 R U5 ^ U3RU5  stranded D M A  «g J^ff^** ™~ u3r  ru5 Kr\/  111 Virion budding and maturation  ^  gag-po!  u 3 r env C J mRNA  Ifl Rev-Independent Cytoplasmic Expression  Iff* Rev-Dependent Cytoplasmic Expression -Rev—1  ftlProviral / < Transcription  4. Integration NUCLEUS  5'LTR  -Tat-  t  4  NF-xB Sp 1 Inducible and Constitutive Host Factors  The virion core is then uncoated to expose a viral nucleoprotein complex, w h i c h contains M A , RT, I N , Vpr, and R N A . Following entry the viral RT and RNase H contained within the complex convert the viral R N A genome to double-stranded D N A . V p r facilitates the nuclear localization of the viral nucleoprotein complex and is especially important for nuclear localization in nondividing cells w i t h an intact nuclear envelope membrane. RT is responsible for the reverse transcription initiated from the 3' end of the host t R N A  L y s  primer annealed to the  primer binding site near the 5' end of the genomic R N A (Figure 5). R T contains an RNase H domain that cleaves the R N A portion of R N A - D N A hybrids generated during the reaction. Following t R N A - p r i m e d initiation, reverse transcription involves two D N A strand transfer reactions that are catalyzed b y RT and are important for the synthesis of both minus and plus strands of the viral D N A . RT has been a major target for drug design. Two classes of RT inhibitors are already i n clinical use: nucleoside analogs such as A Z T (3'-azido2',3'-dideoxythymidine) and d d l (didanosine) that are presumed to bind to the polymerase active sites, and non-nucleoside inhibitors such as nevirapine that binds close to the polymerase active site and thereby keeps RT inactive. Following reverse transcription, I N catalyzes integration of the viral D N A into a host chromosome and the D N A is then repaired.  U p o n cell activation, viral transcripts are expressed from the promoter located i n the 5' long terminal repeats (LTR) (Step 5, Figure 4), with Tat greatly enhancing the rate of transcription. Tat binds to an R N A hairpin k n o w n as the transactivating  response element (TAR) located at the 5' end of the nascent viral  21  Figure 5 H I V - 1 reverse transcription. The viral R N A genome has plus strand polarity and is shown as a thin solid line, designated (+) (Luciw, 1996). Synthesis of the first strand of D N A [negative polarity, represented as a heavy solid line and designated (-)] is primed by a t R N A molecule hydrogen bonded to the PBS of the template R N A . Synthesis of the second D N A strand is primed by a short viral R N A fragment generated by Rnase H activity at the PPT that borders U 3 and a central PPT (cPPT) located i n viral sequences near the 3' end of the p o l gene. The second D N A strand has plus strand polarity and is represented as a heavy gray line. A CTS, w h i c h functions to terminate plus strand viral D N A in step F, is located near the 3' end of the pol gene and is shown as a solid circle; about 100 nucleotides of plus strand D N A is displaced to allow for termination at the CTS. To simplify the pictorial representation of lentivirus reverse transcription, important cis-acting signals (PBS, CTS, cPPT, and PPT) are shown only in key steps. PBS: primer binding site; PPT: polypurine track; cPPT: central polypurine track; CTS: central terminal signal. (Figure reproduced from Luciw, 1996). l y s  22  A. (-) strand priming c  c ^ ' R N A primer  PBS  ^  . cPPT  PPT  T B. first strand-transfer  R^U5 ^ y ?  C. (-) strand elongation ^  U3  R U5  ^V?  Poly (A)  D. fa) strand priming U3  R U5PBS  £. second strand-transfer U3  U3  R  R  U5  U5PBS  < U3  R  F. fa) strand elongation  •  U3  R U5  *y—  U5 Y  G. strand completion  U3  U3  R U5  R  U5  transcripts through its arginine-rich domain, and enhances the efficiency of the transcription polymerase in the production of viral m R N A s up to 100 fold. Tat is therefore essential for viral replication.  Spliced and genomic-length R N A s are then selectively transported b y Rev (Figure 6), from the nucleus to the cytoplasm, where viral m R N A s are translated, viral genomic R N A s are packaged and new virons are assembled. W h e n viral m R N A s are first produced, most are multiply spliced and encode the Tat, Rev, and Nef proteins. Afterwards, singly spliced and unspliced transcripts are transported to the cytoplasm, where viral proteins are translated and viral genomic R N A s are packaged as infectious virions. Rev is critical for this switch of transporting singly spliced and unspliced transcripts instead of multiply spliced viral m R N A to the cytoplasm. This is achieved by a binding interaction between Rev and the Rev responsive element (RRE) site located in the env coding region. V p u promotes degradation of C D 4 which holds newly synthesized Env glycoprotein (gpl60) i n the endoplasmic reticulum, thus allowing E n v transport to the cell surface for assembly into viral particles. In addition to its role i n C D 4 degradation, V p u can also stimulate virion release. Nef, an important regulatory HIV-1 viral protein which w i l l be discussed in more detail later i n this chapter, downregulates C D 4 from the cell surface. By doing so, it may enhance Env incorporation into virions, promote particle release, and possibly affect CD4+ Tcell signaling pathways. Nef can also downregulate the cell surface expression of M H C class I molecules, which may help protect infected cells from killing by cytotoxic T cells. In addition, Nef has been shown to enhance viral infectivity in vitro and to be responsible for disease progression in patients and animal  24  Figure 6 Splicing patterns of HIV-1 transcripts. T h e gag a n d gag-pol m R N A s are u n s p l i c e d ; m R N A s for the early g e n e s tat, rev, a n d nef are doubly s p l i c e d ; m R N A s for the late g e n e s vpu, env, vif, a n d vpr are singly s p l i c e d . A l s o s h o w n are two m R N A s p e c i e s which e n c o d e novel regulatory g e n e s : tev/tnv a n d the first e x o n of tat. S h o w n in p a r e n t h e s e s are additional transcripts g e n e r a t e d by alternative splicing (vpr, m i n i e x o n s 2 a n d 3). (Figure r e p r o d u c e d from Luciw,1996).  v. models. V i f is important for the production of highly infectious virions and may also play a role i n viral assembly and maturation.  M A is the N-terminal component of the Gag polyprotein and is important for targeting G a g and Gag-Pol precursor polyproteins to the plasma membrane prior to viral assembly. In the mature viral particle, the M A protein lines the inner surface of the virion membrane. C A is the second component of the G a g polyprotein and forms the core of the virus particle, w i t h around 2000 molecules per virion. N C is the third component of the Gag polyprotein and coats the genomic R N A inside the virion core. The primary function of N C is to b i n d specifically to the packaging signal and deliver full-length viral R N A s into the assembling virion. P6 comprises the C-terminal 51 amino acids of Gag and is important for incorporation of V p r during viral assembly. The immature virion is composed of cell membrane embedded w i t h Env and Gag viral proteins, and a viral core assembled from the Gag and Gag-Pol polyproteins, accessory proteins, Vif, V p r Nef, and two identical strands of the genomic R N A . A s the particle buds and is released from the cell surface, virion maturation occurs through the proteolytic processing of the Gag and Gag-Pol polyproteins by PR. P R cleaves at several polyprotein sites to produce the final M A , C A , N C and p6 proteins from Gag, as well as PR, RT, and I N proteins from Pol. Because viral assembly and maturation must be highly coordinated, factors that influence P R activity can have dramatic effects on virus production. P R has been a prime target for drug design, and several P R inhibitors are already in clinical use (Hammer, 1996). After the virion maturation step, which involves proteolytic processing of the Gag and Gag-Pol polyproteins by P R and a less well defined function of Vif, the  26  viral replication cycle is completed and the mature virion is then ready to infect the next cell.  1.6 An overview of HIV-1 Nef protein  The nef gene is located at the 3' end of the viral genome, partially overlapping the U 3 region of the 3' L T R (Figure 3). Nef is expressed early i n the H I V replication cycle from multiply spliced m R N A transcripts that encode a 27 k D myristoylated protein that is largely membrane associated (Trono, 1995).  Considerable controversy has surrounded the biological function of the H I V - 1 Nef viral protein. Initial studies suggested that this early nonstructural viral protein functioned as a negative regulatory factor and acted to suppress both viral replication and transcription activity of the HIV-1 L T R (Harris, 1996). It was therefore proposed to play a role i n establishing or maintaining viral latency. However, other investigations failed to confirm these negative effects of Nef (Harris, 1996). It is clear n o w that Nef expression is critical for efficient virus replication under conditions similar to virus replication in vivo.  Several distinct functions of N e f have been well characterized in vitro. First, Nef, i n concert w i t h Env and V p u , downregulates the cell surface expression of C D 4 receptors (Piguet et al, 1999b). Such a function may serve to inhibit superinfection and increase the infectivity of the viruses. Nef also downregulates cell surface M H C - 1 molecules and this may play an important role i n the pathogenesis of HIV-1 infection through immune escape by protecting infected cells from recognition of cytotoxic T lymphocytes (Piguet et al., 1999b). Third, Nef  27  increases viral infectivity at the stage of post-entry of the virus into the cell (Aiken and Trono, 1995; Chowers et al, 1995; Schwartz et al., 1995), w h i c h may be responsible for the high viral load i n animal models infected w i t h Nef+ H I V - 1 . Nef also alters cellular signal transduction and activation pathways w h i c h may be linked to the pathogenesis of HIV-1 infection (Baur et al., 1994; Hanna et al., 1998; Iafrate et al, 1997; Skowronski et al, 1993).  The biological importance of Nef has been demonstrated i n several animal models. Transgenic mice expressing Nef developed severe AIDS-like symptoms, thus indicating that ne/harbors a major disease determinant (Hanna et al, 1998). Expression of the Nef protein in transgenic mice perturbs development of CD4+ T cells , elicits depletion of peripheral CD4+ T cells, and alters T cells activation responses (Hanna et al, 1998; Skowronski et al, 1993). Nef has also been shown to be functional i n enhancing both virus replication and pathogenicity of HIV-1 i n S C I D - H u mice (Aldrovandi and Zack, 1996; Jamieson et al, 1994). In Rhesus monkey models infected w i t h simian immunodeficiency virus (SIV), a close relative of HTV, Nef was found to be critical for high viral load and disease progression (Kestler et al, 1991).  Further evidence for the involvement of Nef i n HIV-1 pathogenicity in vivo comes from the observation of Nef deletions i n several case studies of long-term non-progressor (LTNP) patients (Deacon et al, 1995; Kirchhoff et al, 1995). A l t h o u g h these individuals have been infected for more than 10 years, they remain free from HIV-related disease, and have normal CD4+ lymphocyte counts and l o w viral load.  28  1.7 Structure of Nef protein The structure of N e f has been determined by two recent studies, which examined the nuclear magnetic resonance (NMR) structure of the core of Nef (residues 40-206, w i t h a 150-170 deletion) i n solution (Grzesiek et al, 1996a), and the X-ray crystal structure of the Nef core (residues 54-205) bound to the high affinity R96I mutant of the F y n SH3 domain (Lee et al., 1996). Only the crystal structure of Nef protein w i l l be reviewed in this section, since the general features of the crystal structure of the Nef protein are consistent w i t h the demonstration b y N M R of the presence of two anti-parallel a helices and the anti-parallel nature of the P strands (Lee et al., 1996).  The structure of Nef consists of three layers (Figure 7 and 8) (Lee et al., 1996). The N-terminal region forms an outer layer that consists of a polyproline-II (PP-II) helix (Arg71 to Arg77), which contains the P X X P motif, followed by two antiparallel a helices that pack against a middle layer of four anti-parallel P strands. The C-terminal region consists of two short a helices and extends the other side of the P strands.  Nef contains a highly conserved P X X P motif that is responsible for the tight binding interaction w i t h Src family SH3 domains (Kd = 0.25 u M for H c k and 0.38 u M for F y n mutant [R96I]) (Lee et al, 1995). The Src family kinases have a common modular architecture composed of a conserved catalytic domain and two Src homology domains, SH3 and SH2, that bind to proline-containing sequences and phosphotyrosine, respectively (Cohen et al, 1995; Pawson, 1995).  29  Figure 7 T h e crystal structure of the c o n s e r v e d core of HIV-1 Nef c o m p l e x e d with the S H 3 d o m a i n of F y n (R96I mutant). T h e S H 3 d o m a i n is s h o w n in white; the Nef protein is in gray. T h e R T loop is s o n a m e d b e c a u s e of critical arginine a n d threonine r e s i d u e s in S r c . N e f interacts with the S H 3 v i a a polyproline helix ( P x x P ) a n d a h y d r o p h o b i c pocket f o r m e d by the two anti-parallel a - h e l i c e s that follow the P x x P motif. (Reprint with p e r m i s s i o n , from the A n n u a l R e v i e w of B i o p h y s i c s a n d B i o m o l e c u l a r Structure V o l u m e 2 6 , 1 9 9 7 by A n n u a l R e v i e w s w w w . A n n u a l R e v i e w s . o r g ) .  — 71 i ppn HIVNL43+ HIVBAL1 HIVU4 55 A.C HIVELI D.[ HIVZ6 r HIVANT70 HIVMVP5180 L SIVCPZGAB H1VZ321 HIVMAL HIV2ST HIV2ROD HIV2D20S B.C HIV2EHOA SIVMM251 SIVMN SIVSTM SIVAGM155 SIVSYK B-[  HIV-1  HIV-2  SIV  PVR ?QVP J*PMT PVR ?QVP JRPMT PVR PQVP LiRPMT PVE PQVP L.RPMT PVF PQVP L.RPMT PVA PQVP [jRPMT PVR PQVP LiRPMT PVR PQVP TRPHT PVR PQVP JRPMT PVR PQVE JRPMT PVI PRVE liREMT S\n PKVP jRPMT CKIPIVPljRPMT RVR PGVE JRPMT SVR PKVP liRAMT PVRPRVP^RIIS AVF PRVP jREMT PVRPRVPIRQIT PVqP2TEJL.RTLT  141 HIVNL43+ HIVBAL1 HIVU4 55 HIVELI HIVZ6 HIVANT70 HIVMVP5180 SIVCPZGAB HIVZ321 HIVMAL HIV2ST HIV2ROD HIV2D20S HIV2EH0A SIVMM2 51 SIVMN SIVSTM SIVAGM155 SIVSYK  81 i——I  GW GW GW GW GW GW GW GW GW GW GW GW GW GW GW GW GW GF GW  91  "I  YKAAVDLSHFLKE RSAAIDLSHFFKK YKAAFDLSFFLKE YKEALDLSHFLKE YKLAVDLSHFLKE YKGAFDLSFFLKE FKAAFDLSFFLKE YKAAFDLSHFLKE FKGAFDLSFFLKE YKGAFDLSHFLKE YRLARDMSHLIKE HRLAIDMSHLIKT YKLAVDMSHFIKE FKLAVDMSHFLKE YKLAIDMSHFIKE YKLAVDMSHFIKE YKLAIDLSHFIKS YKLAVDFSHFLKE YKLAIDLSHFIKN  n7T-j>— 151 CYKLVPV CFKLVPV CYKLVPV CYELVPV CFELVPV LFKLVPV LFKLVPV CFKLVPL CFKLVPV CFKLVPM LWKLVPV LWKLVPV LWKLVPV LWKLVPI LWKLVPV LWKLVPV LWKLVPV CFKLVPV LWELVPN  «A  161  101 KGGLEGLIHS KGGLEGLIHS KGGLDGLIHS KGGLEGLIWS KGGLEGLIWS KGGLEGLIYS KGGLDGLIYS KGGLEGLVYS KGGLDGLIYS KGGLDGLVWS KGGLEGLYYS RGGLEGMFYS QGGLEGMYYS KGELEGIFYS KGGLEGIYYS KGGLEGIYYS KGGLEGIYYS KGGLDGIYYS KGGLQGMNYC  171  1  111 «a  I  QRRQDILDLWIYHT QKRQDILDLWVYHT QKRQEILDLWVYHT KKRQEILDLWVYNT KKRQEILDLWVYNT HKRAEILDLWVYNT HKRAEILDLWIYHT RRRQEILDLWVYHT KKRQEILDLWVYHT PKRQEILDLWVYHT DRRRRVLDIYLEKE ERRHKILNIYLEKE ERRHRILDTYFENE ERRHKILDTYLENE ARRHRILDMYLEKE ERRHKILDMYLEKE ERRHRILDMYLEKE DRRNKILNLYALNE EKRDEILHLYLQNE  rj^ 121 QGYFPDWQ QGYFPDWQ QGFFPDWQ QGIFPDWQ QGIFPDWQ QGFFPDWQ QGFFPDWQ QGFFPDWQ QGFFPDWH QGYFPDWQ EGIIGDWQ EGIIADWQ EGIVSGWQ EGIVSGWQ EGIIPDWQ EGIMPDWQ EGIVPDWQ WGIIDDWN HGII.DRI  CJE|> 181  EPDKVEE.ANKGENTSLLHPVSLHGMDDF. .ERE EPEKVEE. ANEGENNCLSHPMSLHGMDDP. .EKE DPAEVEE. ATGGENNSLLHPICQHGVDDE. .EKE DPQEVEE. DTEGETNSLLHPICQHGMEDP. .ERQ DPREVEE. ATEGETNCLLHPVCQHGMEDT. .ERE SEEEAERLGNTCERANLLHPACAHGFEDT. .HKE SAEEAERLGNTNEDASLLHPACNHGAEDA. .HGE TEEQVEQ.ANEGDNNCLLHPICQHGMEDE. . DKE DPREVEE. ANTGENNCLLHPMSQHGMDDD. . ERE SPEEVEE. ANEGENNCLLHPISQHGMEDA. . ERE DVP. . QEG. DDSETHCLVHPAQTSRFDDP. . HGE DVP. .QEG.EDTETHCLVHPAQTSKFDDP. .HGE EVP. .AATREEEETHCLMHPAQISSWDDI. . HGE NM. . . IAEPEDEETHCLVHPAQTSAWDDP. .HEE NVS. . DEAQEDER. HYLMQPAOTSKWDDP. . WGE NVS. . DEAQEGEE. NYLLH PAQTSQWDDP. .WGE DMS. . NEAQEDDGTHYLVH PAQTHOWDDP. .WGE ALH. . EEA. ETCERHCLVHPAQLHEDPDGINHGE . E I . .EGCLEYEEHTLLLHPASGQGSSS. . .MGE  VLEWRFD VLAWKFD VLMWKFD VLKWRFN VLKWRFN ILMWKFD ILKWQFD VLVWRFD VLMWKFD VLKWKFD TLVWRFD TLVWEFD TLIWQFD TLVWQFD VLAWKFD VLVWKFD VLVWKFD ILAWKFD . PHVELQ  E ] 191 SR ss ST SR SR RS RS SR SS SS PT PL SL SL PT PT PL PM PP  LAFH LAFH LALK LAFE LAFE LGNT LGLT LALR LARK LALR LAFS LAYS LAYD LAYD LAYT LAYT LAHT LAVQ PGYT  C $ > —  NYT NYT NYT NYT NYT NYT CYT NYT NYT NYT NYT NYT NYT NYT DYT NYT NYT AWS NYT  131 PGPGV PGPGT PGPGI PGPGI PGPGI PGPGT PGPGP TGPGT PGPGT PGPGI HGPGV HGPGV HGPGI HGPGV SGPGI SGPGP AGPGI KGPGI SGPGT  RVPL RFPL RYPL RYPL RYPL RFPL RFPL RFPL RYPL RFPL RYPK RYPM RYPK RYPK RYPK RYPK RYPK RYPR RYPL  TF TF TF TF TF TF TF CF CF TF FF FF YF FF TF TF QF CF IF  fSPl 201 HV HV HR HK HK HV HI HI HL HR YE YE YV YV YE YE YE YD P.  ARELH AREH. AYELH AREMH AREMH AMITH ALQKH AREQH AREMH AREQH AFIRY AFIRY AFNRF AFSRF AYARY AYIRY AFVRH PSREY GWEMA  PEYFKNC PEFYKD. PEFYKN. PEFYKDC PELFQKD PELFPK. PEYYKD. PEFYKD. PEYYKDC PEEFGYK PEEFGHK PEEFGYQ PEEFGYQ PEELEAS PEEFGSK PEEFGSK FTDLYST RLQLERQ  Figure 8 S e q u e n c e alignment of the Nef c o r e . T h e a m i n o a c i d s e q u e n c e s of the c o n s e r v e d core region of Nef protein from different strains of HIV-1, HIV-2, a n d S I V are s h o w n . R e s i d u e s n u m b e r s a b o v e the s e q u e n c e s c o r r e s p o n d to HIV-1 N L 4 - 3 . T h e s e c o n d a r y structural e l e m e n t s of the N e f c o r e crystal structure are indicated by arrows for strands (labeled P A - p D ) a n d b o x e s for a helices (labeled ocA-aD); the PP-II helix is indicated. A loop c o n n e c t i n g p C a n d pD is disordered in the structure a n d is indicated a s a b r o k e n line. R e s i d u e s involved in the interaction with S H 3 are labeled with a n asterisk a b o v e the s e q u e n c e . ( R e p r i n t with permission from Author, L e e et al., 1996).  31  RT-loop  »  Sem5 EAVA EHDF QAGSPD ELSFKRGN TLKVLN  TFVA IWA Hck LFVA F y n LEVA A b l C r k N YVRA P I 3 K QYRA S P E C LVLA Nck2 PAYV TAKA V a v RVRA GAP s t e 6 QTTA S r c  LYDY LYDY LYDY IiYDF LFDF LYDY LYDY KFNY RYDF ILPY ISDY  ESRTET EAIHHE EARTED VASGDN NGNDEE  DLSFKKGE DLSFQKGD DLSFHKGE TLSITKGE DLPFKKGD DIDLHLGD QEKSPR EVTMKKGD MAERED E L S L I K Q T CARDRS ELSLKEGD TKVPDTDEISFLKGD ENSSNPSFLKFSAGD  RLQIVN QKWLE KFQILN KLRVLG ILRIRD ILTVNK ILTLLN KVIVME IIKILN MFIVHN TIIVIB  n-src loop  KDE DPB NTE GD GE ES SSE GD NGE YNB KPE EQ GSL(14)EIG KD STN KCS DG KKG QQG ELE DG DG VLE  ~ distal loop  WYKAEL WWZJWHS WWKARS WWEARS WCEAQT WWNAED WLNGYN WWKVEV WWRGSY WWRGEI WMWVTN WCDGIC  D GN LSTGQ IiATRK LTTGE KN G SE GK ETTGE ND R N GQ Y GR LR DE SB K  EGFI TGYI EGYI TGYI QGWV RGMI RGDF QGFV VGWF VGWF QGLX RGWF  PSNY PSNY PSNY PSNY PSNY PVPY PGTY PAAY PSNY PANY VEDL PTSC  IRM VAP VAR VAP ITP VEK VEY VKK VTE VEE VEE IDS  TE SD VD VD VN YR IG LD EG DY VG SK  Figure 9 S e q u e n c e alignment of S H 3 d o m a i n s . S e v e r a l S H 3 d o m a i n s are s h o w n , a n d the c o m m o n s e c o n d a r y structural e l e m e n t s are b o x e d . T h e R T loop a n d n-Src loop are s o n a m e d b e c a u s e of critical arginine a n d threonine r e s i d u e s in S r c (the R T loop) a n d a long insertion in the n e u r o n a l form of S r c (n-Src loop). (Reprint with p e r m i s s i o n , from the A n n u a l R e v i e w of B i o p h y s i c s a n d B i o m o l e c u l a r Structure V o l u m e 2 6 , 1997 by A n n u a l R e v i e w s w w w . A n n u a l R e v i e w s . o r g ) .  32  SH3 target peptides adopt a polyproline type II helical conformation, characterized by the sequence P X X P (Kuriyan and C o w b u r n , 1997). The Nef P P II helix (R71-R77) that spans the P X X P motif interacts with the SH3 domain of Fyn (R96I) through its RT loop (between the first and second strands of SH3 domain) and n-Src loop (between the second and third strands of SH3 domain) (Figure 7, 8 and 9) (Lee et al, 1996). The R96I mutation i n the RT loop of the F y n SH3 domain greatly increases its interaction with Nef from l o w affinity ( K d >20 uM) to high affinity ( K d = 0.38 uM), which makes it comparable w i t h that of the wild-type H c k SH3 binding with Nef (Kd = 0.25 uM). Trp-119 of SH3 was found at the binding interface between Nef PP-II helix and F y n (R96I) SH3 (Figure 7 and 9). Although the physiological relevance of the interaction of Nef w i t h SH3 domains is unknown, mutagenesis of Nef showed that the P X X P motif is essential for M H C - 1 downregulation (Le Gall et al., 1997; Mangasarian et al., 1999; Schwartz et al., 1996) and optimal spread of HIV-1 i n primary cell cultures (Saksela et al., 1995), suggesting that the virus has evolved to exploit SH3mediated interactions w i t h cellular proteins to evade the host cellular defence system and increase viral replication.  1.8 Nef downregulation ofCD4 and its role in HIV-1 infection  C D 4 is a membrane glycoprotein that is expressed i n T helper lymphocytes and cells of the macrophage and monocyte lineage (Maddon et al., 1986; M a d d o n et at., 1985). It comprises a large extracellular component made up of four immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail.  33  C D 4 plays a key role i n the maturation and proper function of helper T lymphocytes (reviewed in (Weiss and Liftman, 1994)). C D 4 stablizes the interaction between the antigen-specific T cell receptor (TCR) on helper T cells w i t h the antigen-MHC-II complex on antigen presenting cells (Doyle and Strominger, 1987). Following the antigen activation of T cells, C D 4 recruits the p56Lck protein tyrosine kinase to the vicinity of the TCR, through a cysteinebased binding site for Lck, resulting i n cell proliferation and interleukin-2 production (Acres et al, 1986; Shaw et al, 1989; Turner et al., 1990; Veillette et al, 1988; Veillette et al, 1989). C D 4 activation also exerts TCR-independent influences, including phospholipase C stimulation, C a  2+  signaling and apoptosis  (reviewed i n (Foti et al, 1995)).  D u r i n g HIV-1 infection, C D 4 serves as the primary receptor for the envelope glycoprotein (Env), allowing for the docking of these viruses onto the surface of target cells (Dalgleish et al, 1984; Klatzmann et al, 1984; M a d d o n et al, 1986). This initial event allows an interaction between Env and specific members of the chemokine receptor family, such as C X C R 4 or C C R 5 for H I Y - l (reviewed i n (Liftman, 1998)), w h i c h triggers the fusion of the viral and cellular membrane, and results i n delivering the virion inner components into the cytoplasm.  In HIV-1 infection, Nef downregulates C D 4 i n concert w i t h two additional H I V - 1 gene products, Env and V p u (Chen et al, 1996). However, the mechanisms of C D 4 downregulation induced by the three viral proteins are different. Nef acts on C D 4 molecules that have already reached the cell surface. In contrast, Env and  34  V p u modulate C D 4 along the biosynthetic pathway i n the endoplasmic reticulum (ER).  The C D 4 downregulation i n HIV-1 infected cells might serve to inhibit superinfection and to reduce the envelope-induced cytopathic effect. This w o u l d allow a more efficient production of viral particles i n infected cells and contribute to the maintenance of high viral loads. Alternatively, downregulating this viral receptor may contribute to the infectivity of outgoing virions by preventing the interference of high C D 4 levels on the surface of HIV-producing cells w i t h the particle incorporation and function of the viral envelope (reviewed i n (Piguet et al, 1999b)). The presence of high C D 4 levels on the surface of HIV-producing cells can dramatically interfere w i t h the particle incorporation and function of the viral envelope (Piguet et ah, 1999b). By downregulating the viral receptor, N e f together w i t h V p u and Env efficiently counteract this inhibition, thus preserving the infectiousness of outgoing virions.  Nef downregulates C D 4 molecules by acting as a connector between the receptor and intracellular trafficking pathways (Mangasarian et al., 1997) (Figure 10). Nef associates w i t h the C D 4 cytoplasmic tail by recognizing the dileucinebased motif that also functions as an endocytosis signal (Aiken et al., 1994; Grzesiek et al, 1996a; H u a and Cullen, 1997). Residues i n Nef involved i n contacting C D 4 are clustered i n a proximal flexible loop (WLE57-59) and i n the core domain of the viral protein (G95, G96, L97, R106 and L I 10) (Grzesiek et al, 1996a; Grzesiek et al., 1996b) (Figure 11). The immediate downstream partner of Nef for C D 4 downregulation is the clathrin-associated adaptor protein complex-2  35  Plasma membrane  Figure 10 T h e m e c h a n i s m of C D 4 downregulation induced by Nef. Nef d o w n r e g u l a t e s C D 4 m o l e c u l e s by acting a s a connector b e t w e e n the receptor a n d intracellular trafficking pathway. Nef a s s o c i a t e s with the C D 4 c y t o p l a s m i c tail by recognizing the d i l e u c i n e - b a s e d motif. T h e immediate d o w n s t r e a m partner of Nef for C D 4 downregulation is the clathrin-associated adaptor protein c o m p l e x - 2 ( A P - 2 ) . A P - 2 interacts with Nef through its-C-terminal dileucine-based motif ( L L 1 6 5 ) . In the p r e s e n c e of Nef, internalized C D 4 m o l e c u l e s are targeted to l y s o s o m a l degradation. T h i s is a c h i e v e d by a l y s o s o m a l targeting signal in Nef ( E E 1 5 5 ) that m e d i a t e s the binding of Nef to the (3 subunit of coat protein-l (P-COP-1) in early e n d o s o m e . (Reprint with p e r m i s s i o n , from the Immunological R e v i e w s V o l u m e 168, 1999 by M u n k s g a a d International P u b l i s h e r s Ltd., C o p e n h a g e n , D e n m a r k ) .  myristoylation  X  MGGKWSKRSVSGWPAVRERMRRAEPAAEGVGAVSRDLEKHGAITSSNTAA 26 aA  PPII  NAATCAWLEAQEEEEVGFPVR|PQVP)LiRPMTTYKAAypLSHFLK^KGGLEGL 58  51  |  65  IYSbKRQDILDLWVYin  101  106  PB  PA  « B |  95-97  Mab F14.11  pc  QGYFPDWqNYT|PGPGl[RYPIJTFGW|CFKLVPVrEP  110  PD  aC  aD  EKvT5EANEGENNCLLHPMSQHGMDDPEKE[v'LMWKFq^ 151  155  165  MabAG11, AE6, EH1  EYYKDC 201  206  Figure 11 D o m a i n s of Nef implicated in receptor downregulation. T h e determinants important for C D 4 or M H C - 1 downregulation a r e indicated with black a n d o p e n arrows, respectively. T h e important regions in N e f structure are b o x e d . T h e epitopes that are r e c o g n i z e d by m o n o c l o n a l antibodies (Mab) are a l s o d e n o t e d .  37  (AP-2), a heterotetramer mediates the formation of clathrin-coated pits and targets plasma membrane proteins to early endosomes (Le G a l l et al, 1998; Piguet et al, 1998). A P - 2 interacts with Nef through its C-terminal dileucine-based motif (LL165) (Bresnahan et al, 1998; Greenberg et al, 1998a). In the presence of Nef, internalized C D 4 molecules are targeted to lysosomal degradation. This is achieved by a lysosomal targeting signal i n Nef (EE155) that mediates the binding of Nef to the P subunit of coat protein-I (p -COP-I) i n early endosome (Piguet et al, 1999a).  1.9 Nef downregulation of MHC-1 and its role in HIV evasion of the cellular immune response  After exposure to H I V an infected individual becomes acutely viremic. This phase subsequently resolves coincident w i t h the appearance of anti-HIV cytotoxic T lymphocytes (CTLs) which play an important role i n the control of virus load (Borrow et al, 1994; K o u p et al, 1994). CTLs detect infected cells through the T-cell receptor and C D 8 molecules which recognize foreign peptides assembled i n the groove of M H C class I proteins expressed on the surface of almost all mammalian cells. CTLs are well k n o w n for their ability to lyse cells expressing foreign antigens by the release of granzymes and perforins and by the activation of Fas-mediated killing (Berke, 1995). In addition, activated antiH I V C T L s secrete soluble substances including chemokines that potently inhibit infection by H I V (Wagner et al, 1998; Walker et al, 1986; Yang et al, 1996). Recently, the crucial role of CD8+ T cells have been demonstrated i n controlling simian immunodeficiency virus (SIV) replication i n vivo i n monkeys by two laboratories (Jin et al, 1999; Schmitz et al, 1999).  1  38  D u r i n g HIV-1 infection, the cell surface expression of M H C - 1 is downregulated (Kerkau et al, 1989; Scheppler et al, 1989). M H C - 1 consists of a highly polymorphic, membrane-anchored heavy chain non-covalently associated w i t h (32-microglobulin ((32m) (Ploegh, 1998). M H C - 1 molecules are stably expressed at the cell surface, w i t h only a minor fraction of the molecules being internalized spontaneously i n T cells and i n monocytes/macrophages (Neefjes et al, 1990; Reid and Watts, 1990).  So far, N e f is the only HIV-1 viral protein that has been found to downregulate M H C - 1 i n primary CD4+ T cells (Collins and Baltimore, 1999). In Nef producing cells, M H C - 1 molecules are rapidly endocytosed and directed to lysosomes for degradation (Schwartz et al, 1996). Nef does not affect M H C - 1 synthesis and transport through the E R and cz's-Golgi, but rather misroutes the antigen presenting receptor from both the fran-Golgi network (TGN) and the cell surface towards the endosomal pathway (Le Gall et al, 1998; Schwartz et al, 1996).  W i t h the exception of the N-terminal myristoylation signal sequence which is required for its membrane localization and important for both C D 4 and M H C - 1 downregulation, other determinants of HIV-1 Nef which are distinct are responsible for M H C - 1 downregulation (reviewed i n (Piguet et al, 1999b) (Figure 11). They include a conserved a-helix near the N-terminus of the protein (Mangasarian et al, 1999), an acidic stretch i n the proximal region of HIV-1 Nef EEEE65 (Greenberg et al, 1998a), and the polyproline motif forming the SH3binding surface, PXXP78. Nef appears to induce M H C - 1 endocytosis using a not-  39  w e l l characterized cryptic motif in the cytoplasmic domain of the M H C - 1 molecule. The LL165 dileucine motif which mediates the interaction of HIV-1 Nef w i t h adaptor complexes and is required for accelerated C D 4 endocytosis, is dispensable for M H C - 1 downregulation (Mangasarian et al., 1999).  Nef is able to downregulate exogenously expressed M H C - 1 H L A - A and H L A - B molecules, but not H L A - C (Le Gall et al., 1998) or H L A - E (Collins and Baltimore, 1999). The sequence YSQA323 i n the cytoplasmic tail of M H C - 1 H L A - A and H L A B found to be responsible for Nef action is reminiscent of tyrosine-based motifs that mediate endocytosis and sorting of a number of surface molecules (Marks et al, 1996; Sandoval et al, 1994; Trowbridge et al, 1993). H L A - C , which is unaffected by the viral protein (Le Gall et al, 1998), carries a cysteine instead of tyrosine at position 320 of this cryptic endocytosis motif. The adaptor protein complex-1 (AP-1) may be involved i n M H C - 1 downregulation by Nef, since H L A - A and - B molecules accumulate in a region containing clathrin-coated vesicles budding from the Golgi that contain AP-1 in the presence of Nef (Le G a l l et al, 1998). In addition, Nef was shown to bind w i t h the | i l subunit of AP-1  and  the complete AP-1 complex i n yeast two-hybrid and cell-free assays, respectively (Le G a l l et al, 1998).  Nef-induced M H C - 1 modulation protects HIV-1 infected cells against lysis by virus-specific CTLs in vitro (Collins et al, 1998). The HIV-infected cells escape C T L recognition by reducing the density of antigenic peptide complexed to M H C class I antigen on the cell surface. However, unless H L A - C is expressed, downregulating M H C - 1 normally renders target cells more sensitive to  40  destruction by natural killer (NK) cells (Brutkiewicz and Welsh, 1995; Colonna et al, 1993). The Nef-dependent downregulation of M H C - 1 was demonstrated to be selective on H L A - A and - B antigens but not on H L A - C or - E antigens (Collins et al, 1998). A rationale for the specificity of M H C class I downregulation is that H L A - A and - B are the major M H C class I encoded proteins k n o w n to present antigens to C T L s , while H L A - C and - E can interact with various inhibitory receptors on N K cells and can protect cells against these killer cells (Collins et al, 1998). The selective downregulation of H L A - A and - B , but not - C and -E, induced by Nef might therefore permit infected cells to display limited recognition for C T L s without being exposed to the attack of N K cells.  Based on these findings that Nef-expressing H I V infected cells w i t h l o w levels of M H C - 1 class I antigens have a survival advantage i n the presence of C T L s , it is to be expected that these cells w i l l survive longer in vivo, produce more infectious viral particles, and eventually make the immune system more vulnerable to viral infection leading to more rapid disease progression.  In addition to the effects of Nef, other mechanisms that diminish the anti-HIV C T L response have been established. A growing number of studies suggest that antigenic variation is an important element of H I V immune evasion. Individual patients w i t h immunodominant C T L responses can escape the C T L response by generating antigenic variants not recognized by C T L s (Borrow et al, 1997; Goulder et al, 1997; Phillips et al, 1991). In addition, some individuals w h o mount a C T L response to the immunodominant strain appear to lack the ability to respond to immunogenic variants that arise later (Klenerman et al, 1995;  41  Klenerman and Zinkernagel, 1998; M c A d a m et al, 1995). Furthermore, it has been reported that certain types of peptides that are sequence variants of the cognate peptide antigens recognized by antigen-specific T cell receptors can act as antagonists (Klenerman et al, 1994; Meier et ah, 1995). It is not surprising that H I V employs multiple mechanisms to evade the host's immune system and lead to the almost universal lethality of virus infection i n untreated individuals. Therefore, a greater understanding of precisely how the immune system is weakened by Nef and other factors, as well as how to prevent such damage to the host's immune system w i l l provide ways of designing more effective therapeutics to counter H I V infection.  1.10 The application of intracellular single-chain antibody in HTV research  A n imunoglobulin G (IgG) antibody is a bivalent molecule composed of four chains, two heavy chains and two light chains. The heavy chains have four domains, one variable domain ( V ) and three constant domains (C 1-C 3); the H  H  H  light chains have one variable domain (V ) and one constant domain (V ). The L  H  region that is responsible for antigen-specific binding i n an antibody is composed of V and V domains. The recombinant ScFv is commonly composed of the H  L  basic antigen recognizing V and V domains that are joined by a flexible H  L  polypeptide linker (Bird et al, 1988; Huston et al, 1988). Intracellular ScFvs have been constructed and fused to signal peptide domains that target several different cellular compartments, such as endoplasmic reticulum, nucleus, and mitochondria (Persic et al, 1997), i n order to direct target proteins to different cellular compartments.  42  Single-chain antibodies (ScFvs) were studied for their ability to block Nef functions intracellularly i n this thesis research. ScFv can be constructed from the variable domain sequences of an antibody c D N A by molecular biology techniques. There are two common sources: hybridomas and antibody libraries. Hybridomas producing either murine monoclonal (Biocca et al, 1993; D u a n et ah, 1994a; Richardson et al., 1995), which are most commonly used for constructing ScFv, or human monoclonal antibodies (Marasco et al., 1993) have been the source of the V and V c D N A for that particular antibody. Antibody libraries H  L  have been built using c D N A from mouse spleens (Clackson et al., 1991), human peripheral blood lymphocytes (Marks et al., 1991), and bone marrow from the immunized host (Burton et al., 1991). These antibody fragments are expressed on the surface of bacterial phages, which are able to infect and multiply i n E. coli. Antigen-specific binding phages can be isolated and characterized after several rounds of selection and enrichment steps, making use of the character that an antibody expressing phage carries the particular genotype of that antibody. The ScFvs used i n this thesis were derived from murine hybridomas obtained from our collaborators.  Early studies showed that the heavy and light chain c D N A s of an antibody against alcohol dehydrogenase I ( A D H I) can be expressed i n the cytoplasm of yeast (Saccharomyces cerevisiae), and the heavy and light chain polypeptides so produced could neutralize the enzyme intracellularly (Carlson, 1988). Further studies verified that the assembly of an antibody can take place i n the reducing environment of a mammalian cytoplasm (Biocca et al, 1990). The feasibility of joining heavy chain and light chain variable domains through a synthetic linker  43  and maintaining binding specificity and affinity of the parent antibody was first demonstrated i n Escherichia coli (Bird et al., 1988; Huston et al., 1988). This k i n d of recombinant antibody is termed single-chain antibody (ScFv). Following these discoveries and advances i n the field, intracellular ScFvs were shown to bind the target protein and inhibit its functions i n mammalian cells (Marasco et al., 1993).  In addition to the potential gene therapy application of intracellular antibodies for treatment of HIV-1 infection, single-chain antibodies can also provide a powerful research tool for studying gene products intracellularly. In this thesis, ScFvs are used to study the inhibitory effect on the cell surface C D 4 and M H C - 1 downregulation-induced by Nef functions. If successful, it can provide an additional tool to the intracellular antibody arsenal against HIV-1 w h i c h could potentially be used i n combination gene therapy for the treatment of H I V - 1 infection. This study can also provide important information on the use of Nef as a new drug target for the treatment of HIV-1 infection.  1.11 The interaction of Hck SH3 domain with HIV-1 Nef protein  A novel strategy for blocking Nef functions explored i n this research was the use of a dominant negative form of a tyrosine kinase-Hck, a natural intracellular ligand of Nef viral protein, to block Nef from downregulation of host cell surface receptors. H c k belongs to the Src family non-receptor-type tyrosine kinases that consists of nine members (Src, Lck, Hck, Fyn, Fgr, Yes, Blk, L y n , and Yrk) (Brown and Cooper, 1996) (Figure 12). They share a common structural and regulatory mechanism, but differ in cellular expression and localization. Each of these kinases has a N-terminal signal region for myristoylation. The post-  44  translational modification of these kinases allows them to attach to the cell plasma membrane. Adjacent to the N-terminal region is a Src homology domain (SH3) w h i c h binds to target proteins via specific proline-rich sequences that adopt a polyproline type II helical conformation (Cohen et ah, 1995; Pawson, 1995). C-terminal to the SH3 domain is the SH2 domain w h i c h binds tightly to specific tyrosine-phosphorylated sequences. The SH2 domain is followed by the kinase domain and a C-terminal tail with a highly conserved tyrosine residue essential to kinase regulation. SH2 and SH3 domains cooperate i n the negative regulation of Src family kinase activity. The dominant-negative H c k (DN-Hck) used i n this work contains only the N-terminal myristoylation signal region, and the SH3 and SH2 domain, without the C-terminal kinase domain.  Y416 SH4  g  PXXp  SH3  SH2  CATALYTIC DOMAIN  yristic acid moiety Figure 12 Structure of Src family tyrosine kinases. The functional domains of Src family tyrosine kinases were shown (SH4, SH3, SH2 and the catalytic domain). Phosphorylation at Y527 in the C-terminal regulatory tail inactivates the kinase activity, and phosphorylation at Y416 activates the kinase activity. The intramolecular binding site of the SH3 domain is denoted by the P X X p consensus sequence (the second proline residue is conserved i n only four of the nine family members, including Hck). (Figure reproduced frm Sicheri and Kuriyan, 1997).  45  H c k is expressed primarily i n granulocytes, monocytes, and macrophage (Holtzman et al, 1987; Quintrell et al, 1987; Ziegler et al, 1987). Several lines of evidence suggest that H c k regulates phagocyte differentiation and function. H c k expression is strongly induced by agents that promote macrophage differentiation (Boulet et at., 1992; Lichtenberg et al, 1992). H c k also associates w i t h the Fc receptor and is activated following receptor engagement (Durden et ah, 1995; Ghazizadeh et al, 1994; Wang et al, 1994) which may serve to activate the respiratory burst. In addition, H c k has been implicated i n hematopoietic cytokine signal transduction. In this regard, interleukin-3, granulocytemacrophage colony-stimulating factor, and leukemia inhibitory factor have all been shown to induce H c k kinase activation (Anderson and Jorgensen, 1995; Ernst et al., 1994; Linnekin et al, 1994).  Nef contains a polyproline SH3-binding motif, PXXP78, that directs specific binding to H c k , L y n , and Lck (Collette et al, 1996; Saksela et al., 1995) and is strictly conserved among the different HIV-1, HIV-2 and SIV isolates (Figure 8). Full-length Nef binds the H c k SH3 domain w i t h the highest affinity reported for an SH3-mediated interaction (Kd = 0.25 uM). The significance of the interaction of Nef w i t h non-receptor-type tyrosine kinases has been supported by the finding that disruption of the Nef proline-rich motif impairs the replicative potential of HIV-1 i n cultured peripheral blood mononuclear cells (PBMCs) (Saksela et ah, 1995).  A dominant-negative mutant of a cellular protein kinase has been widely used for dissecting signal transduction pathways (reviewed i n (Perlmutter and  46  Alberola-Ila, 1996)). A n advantage of using the dominant-negative mutant to study signal transduction pathways is that there is no need to k n o w the exact pathways to w h i c h it contributes. In this w o r k , a strategy for blocking Nef functions using a dominant-negative H c k mutant (DN-Hck) was adopted. The rationale for this is that H c k was shown to bind Nef w i t h high affinity through the N e f SH3-binding motif that has been involved i n M H C - 1 downregulation. By using the D N - H c k approach, it is possible that the Nef-SH3-binding domaindependent effect can be blocked.  In addition, the same H c k mutant devoid of the kinase domain has been studied for its effect on HIV-1 infectivity i n target cells (Tokunaga et al., 1998). Interestingly enough, viral particles assembled i n the presence of D N - H c k demonstrated reduced infectivity, suggesting that D N - H c k was inhibiting Nefinduced activation of specific Src kinases at an early post-viral entry stage (Tokunaga et al, 1998). In these studies, it is hypothesized that D N - H c k might also be capable of interfering with Nef-mediated effects within cells that express this viral protein. In keeping w i t h this notion, we found that D N - H c k prevented Nef-induced class I M H C downregulation, and that this effect was dependent on the presence of a functional H c k SH3 domain. The ability to block Nef function using this approach is of potential therapeutic importance, given that an intact Nef SH3-domain binding site is required for the downregulation of M H C - 1 , and the expression of M H C - 1 molecules is essential for CTLs to recognize and eliminate virus infected cells. In addition, since the SH3-binding site has also been shown to play a role i n other viral activities, such as regulation of infectivity, cell  47  activation and abnormalities of cell signal transduction pathways, therapies directed against this Nef site could have additional benefits for the patients.  1.12 Thesis objectives and hypotheses  The specific aims of this thesis were to: 1) genetically engineer single-chain antibodies against Nef viral protein; 2) evaluate the effect of expressing intracellular single-chain antibodies against Nef, monitored by cell surface expression of CD4 and M H C - 1 ; 3) evaluate the effect of expressing a dominantnegative H c k mutant i n cells transfected w i t h Nef, represented by the cell surface expression of CD4 and M H C - 1 .  The hypotheses addressed i n this thesis are: 1) Nef-induced M H C - 1 downregulation enables HIV-infected cells to escape killing by cytotoxic Tlymphocytes, which may be responsible for the failure of the host immune system to control H I V infection and disease progression, 2) intracellular expression of Nef-specific single-chain antibodies or the expression of a dominant-negative form of Hck, a Src-family tyrosine kinase w h i c h has been shown to have high binding affinity with Nef, could potentially inhibit the immune dysfunction induced by Nef i n HIV-infected cells, 3) interfering w i t h Nef-induced class I M H C - 1 downregulation may therefore represent a novel strategy for controlling H I V infection by increasing HIV-specific cytotoxic cell activity against infected cells.  The rationale supporting these proposals are : 1) HIV-specific cytotoxic T lymphocytes (CTL) have been shown to be an important host cellular defence  48  factor against viral infection, since the large increases i n the number of CD8+ T lymphocytes and the appearance of virus-specific CTLs correlates w i t h the decrease of viral load and the resolving of the acute viremia phase of H I V infection into the subsequent period of clinical latency that is variable i n length and proportional to the amount of circulating virus (Borrow et al, 1994; Collins and Baltimore, 1999; K o u p et al, 1994; Pantaleo and Fauci, 1996). A n inverse correlation between the quantity of anti-HIV CTLs and viral load has also been found, i n support of the importance of CTLs in the host defence against H I V infection (Daar et al, 1991). In addition, HIV-specific C T L activity is also correlated to disease progression and strong HIV-specific C T L activity is associated w i t h long-term nonprogression of disease i n a small group of H I V infected individuals who have controlled their viremia in the absence of antiviral therapy (Harrer et al, 1996).  2) The critical role of Nef in the establishment of high viral loads is best demonstrated by the requirement of an intact nef gene for the development of A I D S i n humans and monkeys (Deacon et al, 1995; Kestler et al., 1991; Kirchhoff et al., 1995). Nef is a 27 k D myristoylated protein that mediates C D 4 and M H C - 1 downregulations through different domains. Recent studies have implicated the acidic and proline-rich regions of Nef as being important for M H C - 1 downregulation (Greenberg et al., 1998b). Expression of Nef i n an infected cell was shown to protect it from anti-HIV C T L recognition. This is achieved by downregulation of M H C - 1 i n infected cells and hence to prevent C T L recognition by reducing the density of antigenic peptide complexed to M H C class I antigen on the cell surface (Chen et al, 1996; Collins et al, 1998). It was demonstrated that  49  Nef-mediated M H C class I downregulation can be profound and that Nefexpressing cells w i t h low levels of M H C class I antigens have a survival advantage i n the presence of CTLs (Collins et al, 1998). Thus the activity of Nef is likely to be synergistic with other reported mechanisms of H I V escape from C T L s that rely on antigenic variation (Borrow et al, 1997; Goulder et al, 1997).  3) Intracellular expression of ScFvs can either block function or sequester the protein of interest. ScFvs, w h i c h have been shown to have specific binding affinities equivalent to those of the parent monoclonal antibodies (Whitlow, 1991a; Winter and Milstein, 1991), can be stably expressed intracellularly where they are capable of inactivating specific cellular gene products (Carlson, 1988). Intracellular ScFv proteins with specificity for Nef thus provide a unique way of studying the mechanism of Nef i n virus evasion of the host immune system, as well as offering a potential gene therapy strategy for inhibiting the disease progression i n H I V patients.  Similarly, other Nef-ligands that bind with Nef intracellularly could also potentially block the function of Nef or sequester the Nef protein. D N - H c k , consisting of the H c k amino-terminal domain, and the SH3 and SH2 domains, is a good candidate. Src family protein-tyrosine kinases, including Hck, Lck and L y n , are able to bind the polyproline (PXXP78) motif i n Nef w i t h varying affinities by virtue of their SH3 domain (Lee et al, 1996; Saksela et al, 1995), w i t h H c k SH3 domain has the highest affinity yet reported for an SH3-polyproline tract interaction w i t h Nef protein ( K d = 0.25 uM) (Lee et al, 1995). In addition to the high affinity of the interaction between H c k SH3 domain and Nef, the  50  polyproline binding motif i n Nef has been shown to be critical for class I M H C downregulation induced by Nef expression (Greenberg et al, 1998b). This raises the possibility that D N - H c k expression may inhibit M H C - 1 downregulation induced by Nef.  Current combination therapies aimed to inhibit H I V replication can suppress H I V i n the blood stream of infected patients to such a level that is below the limits of detection using the most sensitive detection method. But they are ineffective i n eradicating infected reservoir cells that persist i n the host immune system and continue to produce low level of virus i n the blood (Baiter, 1997; H o , 1997). Therefore, removing the virus reservoir cells is critical for virus eradication from an infected individual. In this regard, Nef may be a potential drug target for reducing virus infected cells, since one of its detrimental effects enables the infected cells to escape killing by CTLs through downregulation of class I M H C molecules. Thus, therapies directed against Nef could increase the probability of infected cells being recognized and subsequently killed by C T L .  The results presented i n this thesis suggest that intracellular binding to Nef alone, as demonstrated by single-chain antibodies, is not sufficient to inhibit the downregulation of M H C - 1 molecules induced by Nef. Specific binding interaction to the SH3-binding surface i n Nef, such as that of the D N - H c k w i t h Nef, is required for such an action. Chapter 3 addresses the hypothesis that Nefspecific intracellular single-chain antibody (ScFv) expression could potentially inhibit the C D 4 and M H C - 1 downregulation effect induced by Nef. Using c D N A reverse transcribed from m R N A of hybridoma clones, several anti-Nef ScFvs  51  were constructed from the variable regions of monoclonal antibodies that recognized the C-terminus and a central domain (Nef83-88) i n Nef. A l l of them retained the binding activity of their corresponding parental monoclonal antibodies when expressed intracellularly as demonstrated by 'pull-down' assays using recombinant Nef protein. However, none of the ScFvs was able to inhibit C D 4 or M H C - 1 downregulation when co-expressed with Nef i n the in vitro transient transfection system developed i n this thesis. It is concluded that the intracellular binding interaction of ScFv w i t h Nef and the following Nef sequestration may not be sufficient to block the receptor downregulation events induced by Nef. The expression of an intracellular Nef-ligand capable of binding to domains i n Nef that are implicated i n C D 4 or M H C - 1 modulation may be required for this effect.  In Chapter 4, the question of whether expression of a dominant-negative H c k mutant (DN-Hck) can inhibit class I M H C downregulation is addressed. D N - H c k , consisting of the H c k amino-terminal domain, and the SH3 and SH2 domains was co-expressed w i t h Nef i n the transient transfection system as described above. The results suggest that D N - H c k prevented Nef-induced class I M H C downregulation, and this effect was dependent on the presence of a functional H c k SH3 domain. It is concluded that the SH3-binding surface on Nef represents a target for therapeutic intervention in individuals infected w i t h HIV-1. Therefore, interfering w i t h Nef SH3 binding site function, as we have done w i t h D N - H c k , represents a potential therapeutic strategy for assisting the host immune system to eliminate HIV-l-infected cells. The results also suggested a model i n w h i c h D N - H c k prevents Nef-induced class I M H C downregulation by  52  blocking the interaction between Nef and an as yet unidentified SH3-containing cellular protein that is able to couple Nef to the M H C molecule. U p o n binding w i t h Nef, this cellular protein might recruit class I M H C molecules via an interaction w i t h their cytoplasmic tyrosine-based sorting motifs, w h i c h i n turn routes these molecules towards an intracellular degradation pathway (Greenberg et al, 1998b; Le Gall et al., 1998; Mangasarian etal, 1999).  53  CHAPTER 2 MATERIALS A N D METHODS 2.1 Materials Anti-Nef hybridoma clones and monoclonal antibodies A G 1 1 , A E 6 and E H 1 were obtained from D r . James Hoxie, University of Pennsylvania. The clones A G 1 1 and A E 6 were raised against the recombinant Nef protein of H I V - I L A I strain and were derived from the same fusion. They both produce I g G l monoclonal antibodies that recognize Nef of the H I V - I L A I / but not SF2 strain, and are specific for the C terminus of Nef (epitope: V A R E L H P E Y F K N C ) . Clone E H 1 , raised against the Nef protein of the HIV-1SF2 strain, is an I g G l monoclonal antibody that reacts w i t h Nef from both HIV-1 L A I and SF2 strains. It was also mapped to the C-terminus of Nef protein (epitope: M A R E L H P E Y Y K D C ) . A monoclonal antibody clone F 1 4 . l l (a gift from D r . Rita De Santis, Menarini Ricerche Sud, Rome, Italy) generated against a different epitope of Nef (Nef 83-88 A A V D L S ) , was also used (De Santis et ah, 1991). :  P4.2, a H e L a cell line expressing the human C D 4 molecule, was obtained from Dr. Oliver Schwartz, Institut Pasteur. This cell line has been successfully used for studying C D 4 and M H C - 1 receptor downregulation b y transient expression of Nef (Le G a l l et ah, 1998; Schwartz et ah, 1996).  Nef expression vectors: The C M V - d r i v e n Nef-FT vector (pCMV-Nef-FT) carrying the nef LAI gene and the control Nef plasmid carries the ne/gene i n an antisense orientation ( p C M V - A S ) are both from Dr. Oliver Schwartz, Institut Pasteur (Le Gall et ah, 1998).  54  Vectors expressing single-chain antibodies: Single-chain antibody constructs derived from E H 1 and F 1 4 . l l were tagged w i t h myc at their C-termini by subcloning into a mammalian expression vector, pDEF-myc, constructed from pDEF3 (Kozak, 1987). Further modifications were done to fuse the Nef myristoylation signal sequence ( M G G K W S K R S V S G W P A V R E R ) to the N-termini of the ScFv  -  constructs by PCR. The resulting pDEF-ScFv-myc and pDEF-myrScFv-myc vectors have a E F - l a promoter and a myc tag at the C-terminus of the expression cassette (Figure 20).  Expression of dominant-negative Hck and mutants: Expression vectors for the  dominant H c k and its mutants (Figure 23) (Tokunaga et al., 1998) are gifts from Dr. M i c h i y u k i Matsuda, International Medical Center of Japan, Japan. The dominant-negative H c k expression vector, p C A G G S - D N - H c k , consists solely of the amino-terminal regulatory domains, SH3 and SH2. The D N - H c k - W 9 3 F vector has substituted Trp93 w i t h Phe, and the DN-Hck-R151S vector has a A r g l 5 1 to Ser substitution. These two residues are essential for the functions of SH3 and SH2, respectively (Tokunaga et al, 1998). C r k l l adaptor protein, w h i c h consists mostly of the SH2 and SH3 domains, was used as a control for D N - H c k (Tokunaga et al, 1998).  2.2 Inhibition ELISA assay  Recombinant Nef-GST produced from E. coli transformed w i t h pGEX-BHIONef (the plasmid is a gift from D r . Mark Harris, University of Glasgow, U K ) (Harris et al, 1992) was used as the coating antigen. This recombinant Nef protein from H I V -  55  iBHIO shares 96% identity w i t h the amino acid sequence of H I Y - I L A I - Microtiter wells were coated w i t h 50 u l of Nef-GST per well (12.5 f i g / m l i n PBS buffer) overnight at 4 ° C . The wells were blocked w i t h 1% B S A / P B S for 1 hr at 37°C. M a b from clone E H 1 was biotinylated using the ImmunoPure Sulfo N H S - L C Biotinylation K i t (Pierce). Serially-diluted antibodies from clones A G 1 1 and A E 6 , as w e l l as a M a b (F14.ll) generated against a different epitope of Nef (Nef 83-88  :  A A V D L S ) , were mixed w i t h 5 | i g / m l biotinylated EH1 Mab i n the wells and incubated for 1 hr at 37°C. After three washes with 0.05% Tween 20/PBS, avidinalkaline phosphatase (Pierce) was added at a dilution of 1:1000 and incubated at 3 7 ° C for 1 hr and washed as above. Immune complexes were detected by the enzyme-substrate reaction w i t h p-nitrophenyl phosphate (Sigma), w i t h the reactions being read at 405 n m after 30 min.  2.3 RNA isolation and reverse transcription-polymerase chain reaction Total R N A was isolated from 10 hybridoma cells, using the guanidinium 7  thiocyanate method (Chomczynski and Sacchi, 1987). The variable regions of the light chain and heavy chain were amplified by RT-PCR, using the following primers: forward primers: V F o r w a r d (5' A A G C T T C C A T G G A [ C T ] [ A G ] T [ T C ] [ T G ] K  [ T A ] G A T G A C [ C A ] C A [ G A ] [TA]CTCC 3'), VFjForward (5' G G A T C C G G T G G T G GTGGTTCTGGTGGTGGTGGTG[AG]GGT[CG]CA[AG]CT[GT][GC][TA]G[GC]AGT C [ A T ] G G 3'); and reverse primers: V Reverse (5' G G A T C C A C C A C C A C C A T T G A K  T T T C C A G C T T G G T G C C A G C A C C G A A C G 3'), VHReverse (5' A A G C T T C T A T G A G G A G A C G G T G A C C G T G G T C C C G G G G C C C C A G 3') (Figure 12a). Primers VKForward and V n F o r w a r d are degenerate primers w i t h alternative bases  56  indicated i n the brackets. The synthesis of c D N A was performed using Superscript Moloney leukemia virus ( M - M L V ) reverse transcriptase ( G I B C O - B R L / L i f e Technologies) according to manufacture's instructions. The reverse primers were used to prime the reverse transcription reactions. Polymerase chain reactions (PCR) were then performed using Taq D N A polymerase (Perkin-Elmer) i n a G e n e A m p P C R System 9600 (Perkin Elmer), for 35 cycles under the following conditions: denaturation at 96°C for 30 sec, annealing at 58°C for 45 sec, and extension at 72°C for 1 m i n ; finishing w i t h 72°C for 10 m i n .  2.4 Cloning and sequencing of amplified products  Amplified D N A fragments were digested w i t h BamHI and H i n d l l l and gel purified by electrophoresis on 1.5% agarose gels, using the QIAquick gel extraction kit ( Q I A G E N ) . The purified products were ligated into the B a m H I / H i n d f f l restriction sites of a cloning vector, p G E M - 4 Z (Promega), and transformed into competent E. coli D H 1 0 B ( G I B C O / B R L ) . Several recombinant clones were selected and sequenced in both directions using dye-terminator cycle sequencing kit (Applied Biosystems), and T 7 / S P 6 promoter primers (University Core D N A Services, University of Calgary, Canada) on a 373 automated sequencer (Applied Biosystems). Alignment of the antibody sequences was performed using Clustal V (Chomczynski and Sacchi, 1987). Sequences of the recombinant clones were also compared w i t h the non-redundant database at National Center for Biotechnology Information (NCBI) using the Blast program (Altschul et al., 1990).  57  Figure 12a Single-chain antibody construction. Following R N A isolation from hybridoma cells, RT-PCR was performed and the variable regions of the light and heavy chain were cloned and the sequences were analyzed. Single-chain antibodies were constructed by ligating the V and V D N A fragments via the BamHI site at the 3' end of V and 5' end of V . P C R was used to add the Kozak sequence ( G C C A C C ) , the start codon, and the restriction sites, EcoRI and N h e l , to the 5' and 3' ends of the ScFv constructs, respectively. Alternatively, overlapping P C R was performed to introduce the Nef myristoylation signal sequence to the N-termini of ScFv constructs, together w i t h Kozak sequence, the start codon, and restriction sites as above. The resulting ScFv or myristoylation modified-ScFv (myr-ScFv) cassettes were subsequently cloned into a mammalian expression vector that has a EF-lot (elongation factor) promoter and either a G F P reporter or a myc tag at the C terminus of the expression cassette. The predicted molecular weight of expressed proteins are: ScFv-GFP, 54.2 k D ; ScFv-myc, 31.1 k D ; G F P alone, 27.0 k D ; myc alone, 3.8 k D . (Compute p I / M w tool, E x P A S y Molecular Biology Server, Swiss Institute of Bioinformatics, www.expasy.ch/tools/pi_tool.html). k  H  k  H  58  Hybridoma cells (10 ) 7  o  Isolation of RNA  Total RNA RT-PCR  RNA template ^VHReverse  VicReverse  VHForward (BamHI)  VKForward  PCR product VHReverse  VKReverse (BamHI)  Restriction digestion and ligation  BamHI ScFv cassette VK  (GGGGS)3 VH  linker PCR and cloning into mammalian expression vectors  myristoylation sequence-fused ScFv cassette (myr-ScFv)  ScFv cassette  Nef-MF(EcoRI, Kozak sequence, start codon) Forward (EcoRI, Kozak sequence, start codon) Reverse (Nhel)  /  ScFv-MF ScFv-MR (Nhel)  Nef-MR  Start codon  Stop codon  / EF-1oc  ScFv or myr-ScFv cassette  GFP reporter or myc tag  pDEF-ScFv-GFP/pDEF-ScFv-myc/pDEF-myr-ScFc-myc vector  59  )  2.5 Construction and expression of ScFv  The ScFvs were constructed by ligating the V k and V H D N A fragments v i a the B a m H I site at the 3' end of V  K  and 5' end of V H - P C R was used to add the Kozak  translation consensus sequence, G C C A C C (Kozak, 1987), the start codon, and the restriction sites, EcoR I and N h e l I, to the 5' and 3' ends of the ScFv constructs, respectively. The primers used were, Forward primer, G G A A T T C C T G C C A C C A T G G A C A T T T T G A T G A C C C A G T C T ; Reverse primer, C G C C T A G C T A G C T G A G G A G A C G G T G A C C G . P C R products were subsequently cloned into a mammalian expression vector, p D E F - G F P , constructed from p D E F 3 (Kozak, 1987) and pQBI25 (Quantum). The resulting pDEF-ScFv-GFP vector has a E F - l a promoter and G F P reporter gene at the C-terminus of the expression cassette (Figure 12a and Figure 16). H E K 293 cells were transfected w i t h p D E F ScFv-GFP using Superfect (QIAGEN).  For co-transfection studies using both Nef and ScFv vectors, the ScFv cassettes were subcloned into pDEF-myc, a vector w i t h the same promoter, restriction sites and plasmid backbone, but with a myc tag instead of G F P reporter gene (Figure 12a and Figure 20).  For the addition of Nef myristoylation signal sequence to the N-termini of ScFv constructs, the following primers were used: Nef-MF, A C G A A T T C G C C A C C A T G G G T A A G T G G T C A ; Nef-MR, A G A C T G G G T C A T C A A A A T G T C T C T T T C C C T T A C A G C A G G ; ScFv-MF, C C T G C T G T A A G G G A A A G A G A C A T T T T G A T G A C C C A G T C T ; ScFv-MR, T C C A C C G C G G T G G C G G C C G C T C T A G A C T A G C C . Overlapping P C R was  60  performed using N e f - M F / N e f - M R as primers and pGEX-BHIONef as template; as w e l l as S c F v - M F / S c F v - M R as primers and pDEF-ScFv-myc as template, respectively. The two P C R products were annealed and used as a template for primers, N e f - M F / S c F v - M R . P C R conditions were similar as section 2.3. The overlapping P C R products were subsequently cloned into the mammalian expression vector, pDEF-myc (Figure 12a and Figure 20).  2.6 Fluorescent  microscopy  H E K 293 cells were transfected and cultured overnight in chamber slides (Lab-Tek, N U N C ) . The slides were then washed three times with PBS and fixed for 10 m i n i n 4% paraformaldehyde at room temperature. After three PBS washes, the slides were mounted w i t h G e l / T o l Aqueous M o u n t i n g M e d i u m (Immunon, Fisher), and sealed w i t h nail polish. A fluorescent microscope (Zeiss) and C C D camera were used to monitor the G F P fusion protein expression.  2.7 Characterization  of expressed ScFv by  immunoprecipitation  H E K 293 cells were transfected and cultured overnight in 6-well tissue culture plates (Nunclon Surface, N U N C ) , and washed once with cold PBS and lysed i n 1 m l of lysis buffer (0.5% NP-40,100 m M N a C l , 25 m M Tris, pH7.5,2 m M E D T A , 10% glycerol, 50 m M NaF, and 10 M-g/ml of each of the protease inhibitors: leupeptin, aprotinin, soybean trypsin inhibitor). The supernatant was first cleared of cell debris by centrifugation and subsequently pre-cleared by GST cross-linked to Sepharose beads (CNBr-activated Sepharose 4B, Pharmacia). The recombinant NefGST fusion protein cross-linked to Sepharose beads was used to immunoprecipitate ScFv by incubating for 1 hr with rotation at 4°C. The precipitated products were  61  resolved by S D S - P A G E electrophoresis and transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk i n TBS-T buffer (10 m M Tris, 50 m M N a C l , and 0.5% Tween 20) and probed w i t h anti-GFP antibody (1:4000 dilution, Boehringer Manheim). Immunodetection was accomplished using goat anti-mouse antibody conjugated to horseradish peroxidase, washed w i t h TBST and followed by E C L detection (Amersham). The membrane was subsequently exposed to X-ray film ( X - O M A T , Kodak).  2.8 Co-transfection of Nef and ScFv or DN-Hck  P4.2 cells were co-electroporated with 15 ixg of recombinant plasmid expressing Nef (pCMV-Nef-FT) or the antisense control plasmid ( p C M V - A S ) , and 15 jig of the ScFv plasmids (pDEF-ScFv-myc or pDEF-mryScFv-myc), or dominantnegative H c k plasmid ( p C A G G S - D N - H c k ) or its mutants ( p C A G G S - D N - H c k W93F, DN-Hck-R151S), i n association w i t h 4 Lig of the p D E F - G F P reporter plasmid.  Electroporations were performed at 200 V , 960 uE using 4 mm-gap cuvettes i n a Bio-Rad Gene Pulser. Approximately at 20 h post-transfection, the cells were harvested by trypsinization and washed w i t h PBS for further analysis.  2.9 Flow cytometry analysis of CD4 and MHC-1 expression  The cells were stained with either anti-CD4-PE (RPA-T4, Pharmingen) or antiM H C - l - P E (G46-2.6, Pharmingen) for 30 m i n on ice i n F A C S buffer (PBS w i t h 2% FBS). The cells were then washed for three times w i t h F A C S buffer. G F P positive  62  cells were gated and analyzed on FACSort flow cytometer (Becton-Dickenson) using CellQuest software.  2.10 SDS-PAGE  and Western blot analysis of expressed protein  The expression of ScFv, dominant-negative H c k and its mutant, the control C r k l l , Nef and G F P were monitored by S D S - P A G E and Western blot of the total cell lysate. S D S - P A G E was performed on 12.5% polyacrylamide gels under reducing conditions. After protein transfer onto nitrocellular membranes, the blots were probed w i t h individual primary antibodies: anti-c-myc-peroxidase (9E10, Boehringer Mannheim), anti-Hck (N-30, Santa Cruz), anti-Crk (Transduction labs), Rabbit anti-Nef serum (331, N I H AIDS Research and Reference Reagent Program) or anti-GFP M a b (Boehringer Mannheim), separately, followed by a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, or anti-mouse, D A K O ) and E C L detection (Amersham).  2.11 Immunoprecipitation of DN-Hck by immobilized Nef-GST fusion protein  For immunoprecipitation studies, cells were lysed in the lysis buffer (0.5% NP-40, 100 m M N a C l , 25 m M Tris, p H 7.5,2 m M E D T A , 10% glycerol, 50 m M NaF, and 10 u.g/ml of each of the protease inhibitors: leupeptin, aprotinin, soybean trypsin inhibitor). The supernatant was first cleared of cell debris by centrifugation and subsequently pre-cleared by GST cross-linked to Sepharose beads. The Nef-GST fusion protein cross-linked beads were used to immunoprecipitate dominantnegative H c k and its mutants. The precipitated products were resolved by SDSP A G E electrophoresis and transferred onto a nitrocellulose membrane. The  63  membrane was then blocked w i t h 2% skim milk i n TBS-T buffer and probed w i t h anti-Hck antibody (1:4000 dilution, Santa Cruz) or anti-Crk (1:8000 dilution, Transduction Labs) followed by immunodetection as described before.  64  CHAPTER 3 INTRACELLULAR SINGLE-CHAIN ANTIBODIES TARGETING NEF H A V E N O EFFECT O N NEF-INDUCED CD4 A N D MHC-1 D O W N R E G U L A T I O N 3.1 Introduction It is w e l l established that Nef is essential for rapid lentivirus replication in vitro and efficient viral growth i n SIV animal models and human HIV-1 infection (Deacon et al, 1995; Kestler et al, 1991; Kirchhoff et al, 1995). In general Nef has been shown in vitro to: downregulate cell surface C D 4 and M H C - 1 expression (Piguet et al, 1999b); increase viral infectivity at an early post-entry stage (Aiken and Trono, 1995); and disturb cell signaling and activation pathways (Baur et al, 1994; Hanna et al, 1998; Iafrate et al, 1997; Skowronski et al, 1993).  The major interest of this thesis research is to study how to prevent or inhibit the detrimental effects Nef has on host cells. More specifically, the studies i n this chapter examine the possibility of expressing intracellular single-chain antibodies (ScFvs) to block the downregulation of C D 4 and M H C - 1 molecules-induced by Nef. Both of these receptor down-modulation effects may contribute to the H I V pathogenesis. Nef downregulates C D 4 with two other viral proteins, Env and V p u . This may serve to dysregulate CD4+ T cell function, inhibit viral superinfection, and increase viral infectivity (Piguet et al, 1999b). The function of Nef-induced M H C - 1 downregulation may be to prevent cytotoxic T-lymphocyte recognition and the subsequent killing of virally infected cells (Collins et al, 1998).  65  The emphasis of current anti-HIV therapy has been on the inhibition of H I V viral replication by administration of inhibitors for H I V reverse transcriptase and protease. The problem that arises is that, despite efficient viral growth inhibition by the powerful multi-drug "cocktail-therapy", the H I V infection i n a patient can not be eradicated even after several years of treatment. Therefore, therapeutic strategies toward other viral targets need to be studied for their effects on inhibition of H I V infection, as well as on the enhancement of the host immunity to eliminate H I V infection.  The studies i n this chapter examine the effect of Nef-specific single-chain antibody expression on the C D 4 and M H C - 1 downregulation induced by Nef. The rationale for these studies is based on the observation that ScFvs have specific binding affinities equivalent to those of the parent monoclonal antibodies (Whitlow, 1991b; Winter and Milstein, 1991), and can be stably expressed intracellularly where they are capable of inactivating specific cellular gene products (Biocca et al., 1990; Carlson, 1988). Intracellular ScFv proteins w i t h specificity for Nef thus provide a unique way of studying this viral protein as a potential therapeutic target (Marasco et al, 1993), as well as offering a gene therapy strategy for inhibiting the development of AIDS.  3.2 Results 3.2.1 Monoclonal antibodies AG 11 andAE6 bind to an overlapping epitope with EH1.  U s i n g the binding inhibition ELISA assay, it was shown that Mabs from clones A G 1 1 and A E 6 inhibited the binding of the biotinylated E H 1 M a b to immobilized Nef protein (Chang et al., 1998). This binding inhibition was specific, since  66  another M a b , F 1 4 . l l that was mapped to a different epitope failed to inhibit the binding of biotinylated E H 1 M a b to immobilized Nef (Figure 13).  3.2.2 Cloning and sequencing of mouse IgG variable regions  The variable regions of light and heavy chains were cloned from the mouse antiNef monoclonal antibody producing hybridoma clones, A G 1 1 , A E 6 and E H 1 . A t least three clones from two independent RT-PCR reactions were sequenced to minimize the possibility of errors introduced during the amplification step.  A n aberrant  transcript was identified i n all three hybridoma cell lines as  described (Carroll et al., 1988; Duan and Pomerantz, 1994). Using primers specific for the C D R - 1 and CDR-3 regions of the Sp2/0 endogenous kappa chain variable region, it was possible to identify plasmids containing the aberrant kappa chain c D N A using the colony P C R method (Duan and Pomerantz, 1994). A n internal Hindlll  site was found i n the P C R amplified kappa chain (between CDR-2 and C D R -  3, at position 206, Figure 14-A.) from clone E H 1 . Therefore, the T A cloning vector (Invitrogen), instead of p G E M - 4 Z , was used to subclone this c D N A .  Sequences from the three hybridoma cell lines were then analyzed. The aligned D N A and predicted amino acid sequence of the light chain and heavy chain variable regions are shown i n Figure 14 and 15, respectively. The complementarity determining regions (CDRs) were defined according to the Kabat-Wu numbering scheme (Kabat, 1991).  67  Monoclonal antibody concentration (ug/ml) Figure 13 Binding inhibition of biotinylated E H 1 M a b by u n l a b e l e d A G 1 1 ( • ) a n d A E 6 (A) M a b s . F14.11 ( O ) w a s u s e d a s a control M a b . E a c h D a t a s e t is representative of three independent experiment.  68  Figure 14 Alignment of the cDNA sequences of variable regions derived from clones AG11, AE6 and EH1. A : Light chain variable regions. B: Heavy chain variable regions.'-' denotes identical residues;'/' denotes gaps. O n the light chain variable region of clone E H 1 , there is an internal Hindlll restriction site (position 206) (underlined). The complementarity determining regions are indicated.  69  A. AG11  GAC ATT TTG ATG ACC CAG TCT CCA TCC TCC CTA GCT GTG TCA GTT GGA GAG AAG GTT  AE6 EH1  —T  A-G TA- -CA — T C-A  GA — C  AG 11 AE6 EH1  CDR1 TCT ATG AAC TGC AAG TCC AGT CAG AAC CTT TTA TAT AGT AGC AAT CAA AAG AAC TAC A— -GG— A C -CT G-G -A G — A — /// /// /// /// /// /// — A -C T  AG11 AE6 EH1  TTG GCC TGG TAC CAG CAG AAA CCA GGG CAG TCT CCT AAA GTG CTG ATT TAC TGG GCA — C — A AA-T- — A A-A — G ACC — C — T C-T  .. AG11  AE6  EHl  AG11 AE6 EHl  CDR2 TCC ACT AGG GAA TCT GGG GTC CCT GAT CGC TTC ACA GGC AGT GGA TCT GGG ACA GAT AA- -GA TT-  -T- GA-  — A TC- A-G  -GT  — A CA- -C-  TTC ACT CTC ACC ATC AGC AGT GTG AAG GCT GAA GAC CTG GCA GTT TAT TAT TGT CAG A— —C —A -AT T — — C C — G— CA— T A — -G- A — -TA CDR3  AG11  CAA AAT TAT CTC TAT CCT CGG ACG TTC GGT GCT GGC ACC AAG CTG GAA ATC AAT  EHl  T — G— GAG CT- — G T —  B. AG11 AE6 EHl AG11 AE6 EHl AG11 AE6 EHl  GAG GTC CAG CTG GTG GAG TCA GGG CCT GAG GTG GTG AGG CCT GGG GTC TCA GTG AAG — A G—  C — A-- -A-C CDR1 ATT TCC TGC AAG GGT TCC GGC TAC ACA TTC ACT GAT TAT ACT ATA CAC TGG GTG AAG G G —A -CG A-T A-C — C TGG G-G —A CDR2 CAG AGT CAT GCA AAG AGT CTA GAG TGG ATT GGA GTT ATT AGT ACT TAC AAT GGT AAT G— — G -C- -G- C-T G-C — T  G—  -AG  TTA C — GGA -G-  CG-  AG11 AE6 EHl  ACA AAC TAC AAC CAG AAG TTT AAG GAC AAG GCC ACA ATG ACT GTA GAC AAA TCC TCC —T -G— T T— — T G— —C -GT-C -C- — T -C-  AGll AE6 EHl  AGC ACA GCC TAT ATG GAA CTT GCC AGA TTG ACA TCT GAG GAT TCT GCC ATC TAT TAC  AGll AE6 EHl AGll AE6 EHl  -A  C  C — T-C AG  C C-A  A —C  G--  T  CDR3' TGT GCA AGA CCC CTT TAC TAC GAT ACT AAC TAC AGG GAA ATG GAC TAC TGG GGC CCC --T — -AG T-T GGG -CG — T CC- CT- /// /// /// /// /// C GGG ACC ACG GTC ACC GTC TCC TCA  70  A. AG11 EH1  CDR1 DILMTQSPSSLAVSVGEPCVSMNCKSSQNLLYSSNQKNYLAWYQQKPGQSP MYA-L--R-TIT--AN-/ / / / / /DI-T--N-F  K--  AG11 AE6 EH1  CDR2 CDR3 KVLIYWASTRESGVPDRFTGSGSGTDFTLTIS SVKAEDLAVYYCQQNYLY 1 Y-S-T R-NRLVD S--S QAYS LEH--MGI L-YDEL  AG11 AE6 EH1  PRTFGAGTKLEIN G -W  AG 11 AE6 EH1  CDR1 EVQLVESGPEWRPGVSVKISCKGSGYTFTDYTIHWVKQSHAKSLEWIGV AM A-LMK—A AT N-W-E ERPGHG V-E  AG 11 AE6 EH1  CDR2 ISTYNGNTKTYNQKFKDr^TMTVDKSSSTAYMELARLTSEDSAIYYCARPL G -LPGS-R-Y- -E G F-A-T--N QFSS V KSG  B.  AG11 AE6 EH1  CDR3 YYDTNYREMDYWGPGTTVTVSS SYP/////L-S  Figure 15 Deduced amino acid sequence alignment of the variable regions of clones A G 1 1 , A E 6 and E H 1 . A : Light chain variable regions. B: H e a v y C h a i n variable regions.'-' denotes identical residues;'/' denotes gaps.  71  Table 2 Comparison of the variable region sequences of clones AE6 and E H l with A G l l .  % of similarity relative to clone A G l l 1  Nucleic acid sequence Antibody  V  AE6  96.5%  98.6% 95.1%  93.8% 97.5%  91.2%  EHl  73.8%  76.6% 57.9%  61.7% 63.2%  36.8%  K  V  H  Total C D R s  A m i n o acid sequence 2  V  K  V  H  Total C D R s  The similarity study was done according to the overlapping sequences. Total C D R s : The total complementarity determining regions of both V and V .  lr 2  K  H  A l l of the sequences of the variable regions from the three hybridomas contained open reading frames. A t the c D N A level, the total C D R s of clone A G l l was 95.1% similar to that of clone A E 6 (Table 2). Both antibodies recognize the C-terminus of Nef from L A I strain. The total C D R s of clone A G l l , i n contrast, was only 57.9% similar to clone E H l , w h i c h recognizes an overlapping epitope at the C-terminus of Nef (Table 2). A l o w percentage of sequence similarity was also found at the amino acid level when clones A G l l and E H l were compared (Table 2). Thus, while there was 91.2% similarity when the amino acids of the total C D R s of clones A G l l and A E 6 were compared, there was only 36.8% identity when clone A G l l was compared w i t h E H l .  3.2.3 Construction and expression of intracellular ScFv tagged with a GFP reporter Single-chain antibodies (ScFvs) were constructed for clones A G l l and E H l as described i n the Materials and Methods section. The c D N A of the light chain  72  variable region was tethered to the heavy chain variable region through a linker D N A sequence encoding (GGGGS)3. The ScFv c D N A constructs were then ligated to the EcoRI and Nhel sites of the p D E F - G F P expression vector (Figure 16).  To assess the intracellularly expressed single-chain antibodies, H E K 293 cells were transfected and cultured overnight. The transfected cells were then examined using a fluorescent microscope and attached C C D camera. Comparable levels of expression were achieved using all ScFv-GFP constructs, as well as the control vector expressing only G F P (Figure 17).  The ability of the intracellularly expressed ScFv to bind w i t h Nef protein was assessed b y immunoprecipitation of ScFv-GFP, performed using recombinant N e f protein-immobilized on Sepharose beads. The results demonstrated that the immobilized Nef was able to precipitate expressed ScFv-GFP (Figure 18, panel A ) , but not G F P alone (Figure 18, panels B and C). The ScFv constructs were used i n the subsequent intracellular gene expression assays against Nef protein.  73  B.  EF-1cc  GFP  EF-1a  ScFv  Neo  GFP  Neo  Figure 16 T h e single chain antibody a s s e m b l y in p D E F - G F P e x p r e s s i o n vector. A : p D E F - G F P vector which e x p r e s s G F P . B: p D E F - S c F v - G F P vector w h i c h e x p r e s s single chain a n t i b o d y - G F P fusion protein. T h e predicted m o l e c u l a r weight of e x p r e s s e d proteins are: S c F v - G F P , 54.2 k D ; G F P a l o n e , 2 7 . 0 k D ; ( C o m p u t e p l / M w tool, E x P A S y M o l e c u l a r Biology Server, S w i s s Institute of Bioinformatics, www.expasy.ch/tools/pi_tool.html).  Figure 17 H E K 293 cells transfected with pDEF-GFP or pDEF-ScFv-GFP. A : G F P (phase); B: G F P (fluorescence); C: A G l l ScFv tagged w i t h G F P (phase); D : A G l l ScFv tagged w i t h G F P (fluorescence); E: E H l ScFv tagged w i t h G F P (phase); F: E H l ScFv tagged w i t h G F P (fluorescence); G : F 1 4 . l l ScFv tagged w i t h G F P (phase); H : F 1 4 . l l ScFv tagged with G F P (fluorescence). Magnification, x36.  A.  B.  1  1  2  2  3  4  C.  1  5  2  Figure 18 Intracellularly expressed ScFv is immunoprecipitated by immobilized recombinant Nef-GST protein. A, Immunoprecipitation of ScFv with Nef-GST cross-linked to Sepharose beads from cells transfected with the control vector (1), pDEF-GFP (2), pDEF-(AG11)ScFv-GFP (3), pDEF-(EH1)ScFv-GFP (4), pDEF-(F14.11)ScFv-GFP (5). B, Immunoblot of the total cell lysate of the cells transfected with control vector (1) and pDEF-GFP (2). C, Immunoprecipitation of cells expressing the control vector (1) and pDEF-GFP (2) corresponding to B, by Nef-GST cross-linked with Sepharose beads. SDS-PAGE was performed in 12.5% polyacrylamide gels under reducing conditions.  3.2.4 CD4 and MHC-1 downregulation induced by Nef expression in P4.2 cells  To verify that HIV-1 Nef protein is capable of downregulating C D 4 and M H C - 1 receptors (Le G a l l et al, 1998), as well as to identify an optimal dose of Nefexpressing plasmids for the subsequent transfection studies, P4.2, a Hela cell line expressing C D 4 , was transfected with various doses (2-15 |ig) of p C M V - N e f - F T (expressing Nef), or 12 fig of control vector-pCMV-AS (carrying anti-sense Nef sequence), and co-transfected w i t h 4 u.g of p D E F - G F P (expressing green fluorescence protein) as an expression reporter. Flow cytometry was performed for the analysis of C D 4 and M H C - 1 receptors on cells gated for G F P expression 20 hr post-transfection. The extent of receptor downregulation was examined for various levels of Nef expression (Figure 19).  M a x i m u m modulation of M H C - 1 (24-26%) was reached w i t h transfection of 12-15 | i g Nef-FT plasmid. More dramatic downregulation of C D 4 (46-49%) was observed w i t h transfection of Nef-FT plasmid even at low doses (2-4 |ig). Western blot analysis showed that levels of Nef expression were proportional to the amount of transfected Nef-FT plasmid (Figure 19 B). The optimal dose of Nef plasmid D N A was determined to be at 12-15 | i g for transfection assays i n order to accommodate the moderate effect of Nef transfection on M H C - 1 downregulation i n P4.2 cells.  3.2.5 Intracellular expression of ScFv EH1 and F14.ll  did not affect receptor modulation  induced by Nef  Intracellular expression of ScFv against Nef was achieved using two ScFv constructs, E H 1 and F 1 4 . l l (Figure 20). The ScFv F 1 4 . l l that was raised against a central domain of Nef (AAVDLS88) was constructed and its Nef binding capability  78  Figure 19 C D 4 and M H C - 1 modulation i n response to Nef. P4.2 cells were co-electroporated with 4 fig of p D E F - G F P , and the indicated amounts of p C M V - N e f - F T . A . Cells were stained 20 hr later w i t h the a n t i - H L A - A , B, - C M a b or anti-CD4 M a b labeled w i t h P E and analysed by flow cytometry. The contour plots represent C D 4 or M H C - 1 fluorescence levels i n GFP+ cells when the Nef-FT vector used for transfection was 0 (a and g), 2 | i g (b and h), 4 itg (c and i), 8 u.g (d and j), 12 u.g (e and k), and 15 (ig (f and 1). The percentage of cells located i n the lower left quadrant of each plot was indicated. B. Western blot analysis. Lysates from transfected cells were analyzed w i t h a rabbit polyclonal anti-Nef antibody. N T , nontransfected control cells. A S , cells were transfected with 12 u.g of the antisense Nef vector. S D S - P A G E was performed in 12.5% polyacrylamide gel under reducing conditions. Each data set is the representative of three independent experiments.  79  •  F i g u r e 2 0 T h e single-chain antibody a s s e m b l y in p D E F - m y c e x p r e s s i o n vector. A . p D E F - m y c vector which contain the m y c tag. B. p D E F - S c F v - m y c w h i c h e x p r e s s s i n g l e - c h a i n antibody t a g g e d with m y c . C . p D E F - m y r - S c F v - m y c w h i c h e x p r e s s s i n g l e - c h a i n antibody f u s e d with Nef myristoylation signal s e q u e n c e at N-terminus a n d t a g g e d with m y c at C-terminus. T h e predicted m o l e c u l a r weight of e x p r e s s e d proteins are: S c F v - m y c , 31.1 k D ; m y r - S c F v - m y c , 3 3 . 5 k D ; m y c a l o n e , 3.8 k D . ( C o m p u t e p l / M w tool, E x P A S y M o l e c u l a r Biology Server, S w i s s Institute of Bioinformatics, www.expasy.ch/tools/pi_tool.html).  confirmed by immunoprecipitation as described previously. To improve the efficiency of ScFv expression, both ScFv constructs were subcloned into another p D E F vector w i t h a myc tag. Further modification was done to fuse the myristoylation signal sequence of Nef to the N-termini of the two ScFv constructs by P C R as described i n Materials and Methods (Figure 12a and Figure 20). These modifications could potentially serve to increase the efficiency of binding association of the ScFv proteins with Nef since both have membrane anchoring myristoylation signals.  To study the effects of expressing these intracellular antibodies on class I M H C and C D 4 expression i n Nef expressing cells, P4.2 cells were transfected with control vectors, Nef alone, or Nef plus each of the ScFv constructs. Comparable expression levels of E H l , F 1 4 . l l and their myristoylation modified counterparts were obtained in the transient expression experiments (Figure 21). Although Nef expression levels were similar i n the transient transfection of Nef plasmid alone, and Nef plus ScFv E H l or ScFv F 1 4 . l l , the transfection of the two myristoylated ScFv plasmids tended to be associated w i t h more significant reduction i n the levels of Nef expression compared to the transfection of their corresponding non-myristoylated ScFv constructs (Figure 21). This might indicate that the addition of the myristoylation signal sequence to the ScFvs made them more efficient i n reducing the Nef expression level, probably by increasing protein sequestration due to more efficient intracellular binding interaction. A s an expression control, G F P expression levels were also monitored, they were similar i n all transfections w i t h various vectors (Figure 21).  82  Figure 21 C o - e x p r e s s i o n of single-chain antibodies a n d Nef in P 4 . 2 c e l l s . C e l l s w e r e electroporated with 15 ug of p C M V - A S a n d p D E F - m y c (Control), 15 ug p C M V - N E F - F T together with p D E F - m y c vector (Nef), o r p C M V - N E F - F T with p D E F - E H 1 - m y c (Nef+EH1 lane), p D E F - m y r - E H 1 - m y c (Nef+myr-EH1), p D E F - F 1 4 . 1 1 - m y c (Nef+F14.11), p D E F - m y r - F 1 4 . 1 1 - m y c (Nef+myr-F14.11), along with 4 ug of the p D E F - G F P reporter p l a s m i d in e a c h c a s e . C e l l lysates were collected 2 0 hr post-transfection a n d a n a l y z e d by immunoblotting with a m o n o c l o n a l anti-myc antibody ( 9 E 1 0 ) , rabbit anti-Nef antibody a n d m o n o c l o n a l antibodies against G F P , after stripping of e a c h p r e v i o u s blot. S D S P A G E w a s performed in 1 2 . 5 % polyacrylamide gel under reducing conditions.  83  F l o w cytometry of P4.2 cells at 20 hr post-cotransfection w i t h ScFv and Nef expression plasmids demonstrated that the intracellular antibodies expression d i d not prevent C D 4 or M H C - 1 downregulation (Figure 22). The myristoylation modified ScFvs that were shown to reduce Nef expression levels d i d not have significant effect on Nef-induced C D 4 or M H C - 1 downregulation, either (Figure 22). This indicated that the intracellular binding of ScFv w i t h Nef and the Nef sequestration by the transfection of ScFv constructs may not be sufficient to prevent the receptor downregulation induced by Nef. The expression of a molecule capable of binding to the domains of Nef that are implicated i n receptor downregulation may be required for this effect.  3.3 Discussion In this chapter, intracellular single-chain antibody approach was studied to inhibit the cell surface C D 4 and M H C - 1 molecules downregulation induced by Nef, i n order to restore T cell function and to enhance the host cellular immunity to better recognize and clear the infected cells. The knowledge obtained from these studies can be potentially used for the discovery of new drug targets and for the development of alternative gene therapy against H I V infection.  To interfere w i t h Nef function intracellulary, single-chain antibodies against Nef were first constructed and used in the subsequent studies to evaluate their effect on the C D 4 and M H C - 1 receptors downregulation induced by Nef. D u r i n g the process of constructing single-chain antibodies, the sequences of the variable regions from a group of three monoclonal antibodies (AG11, A E 6 and EH1) that recognize an overlapping epitope of Nef protein were cloned and analyzed. It  84  Figure 22 Flow cytometry analysis of CD4 and MHC-1 receptor expression in P4.2 cells co-transfected with Nef and ScFv constructs. Surface C D 4 (a-e) and M H C - 1 receptor (f-j) levels were assessed by F A C S o r t 20 hr post transfection of P4.2 cells with: Nef and pDEF-myc control vector (a and f), N e f w i t h p D E F - E H l - m y c (b and g), Nef with p D E F - m y r - E H l - m y c (c and h), N e f w i t h pDEF-F14.11-myc (d and i), Nef and pDEF-myr-F14.11-rnyc (e and j) (open histograms). The filled histograms i n the top two panels (a and f) represent the NefA S and pDEF-myc control vector-transfected cells. The filled histograms i n others (b-d and g-j) represent the Nef-FT and pDEF-myc vector-transfected cells. These results were representative of three independent experiments.  85  86  has been shown that clones AG11 and A E 6 , but not E H 1 , were highly related. Although the variable region sequences of E H 1 clone were significantly different from those of the other two Mabs, E H 1 recognizes an overlapping epitope within the binding site of the AG11 and A E 6 Mabs. This is supported by the binding inhibition assay which demonstrated that the antibodies from clones A G 1 1 and A E 6 inhibited the binding of the biotin-labeled E H 1 M a b to Nef protein (Figure 13). This study confirmed observations made by others that the antigen recognition capability of an antibody is determined by the protein structure rather than the primary sequence of the antibody variable regions (Mariuzza et al., 1987). More detailed analysis using mutation studies or comparison of crystal structures of the antibody-antigen complex w o u l d be required to elucidate this further.  A strategy for efficient construction and evaluation of single-chain antibodies was developed. The single-chain antibody cassettes composed of c D N A of the variable regions of heavy and light chains that were linked by a D N A sequence encoding a 15 amino acid peptide (GGGGS) were cloned into a plasmid vector 3  w i t h E F a promoter and green fluorescent protein (GFP) fusion at the C-terminus of the ScFv constructs. A transient transfection system using HEK293 cell line was established. G F P was used as a reporter for monitoring ScFv expression i n eukaryotic cells. The advantage of G F P is that the ScFv expression can be readily monitored i n either live or fixed cells. Several anti-Nef single chain antibodies were constructed from the variable regions of monoclonal antibodies that recognize the C-terminus and a central domain (AAVDLS88) i n Nef (Figure 16 and Figure 20). A l l retained the binding activity of their corresponding parental  87  monoclonal antibodies when expressed intracellularly as demonstrated by ' p u l l d o w n ' assays using recombinant Nef protein (Figure 18).  P4.2, a Hela cell line stably expressing C D 4 receptor, was used for studying intracellular single-chain antibody expression against Nef. The effect of intracellular ScFv expression against Nef was studied using two ScFv constructs, E H l and F 1 4 . l l , that recognized the C-terminus and the central domain of Nef ( A A V D L S 8 8 ) , respectively. Both were confirmed for Nef binding ability by immunoprecipitation as introduced previously.  When the ScFvs and Nef are co-expressed i n P4.2 cells after transient transfection, it was somewhat surprising, to find that expression of the intracellular antibody did not significantly inhibit C D 4 or M H C - 1 downregulation induced by Nef. This was unexpected as the ScFvs were shown to b i n d w i t h Nef, and one of the possible modes of action by these intracellular antibodies is to facilitate the sequestration of the Nef protein upon binding. To rule out the possibility of inefficient binding interaction between these two ScFvs and N e f due to post-translational myristoylation of Nef, which makes Nef a membrane-anchoring protein, the two ScFv constructs were further modified to fuse w i t h the Nef-myristoylation signal sequence at the N-terminus. Transient transfection of P4.2 cells w i t h the corresponding N-terminus myristoylationmodified ScFv vectors showed more significant reduction i n the levels of Nef expression compared to the transfection of their corresponding nonmyristoylated ScFv clones (Figure 21). This indicated that the addition of the myristoylation signal sequence to the ScFv clones made them more efficient i n  88  reducing Nef expression level, possibly due to more efficient intracellular interaction w i t h their target protein. However, regardless of the N-terminal myristoylation status, none of the expressed ScFv was able to disrupt C D 4 or M H C - 1 downregulation induced by Nef (Figure 22). This indicated that the intracellular binding of ScFv with Nef and the subsequent Nef sequestration may not be sufficient to prevent receptor downregulation events induced by Nef. The expression of molecules capable of binding to domains i n Nef that are implicated specifically i n receptor modulation may be required for this effect. This confirmed the observation made by others that binding activity of ScFv w i t h a target viral protein can not be used to predict its intracellular activity, and the targeting epitope of an intracellular antibody plays a pivotal role i n disrupting the function of that viral protein (Rondon and Marasco, 1997).  In summary, anti-Nef single chain antibodies were successfully constructed. A l l retained the binding activity of their corresponding parental monoclonal antibodies when expressed intracellularly. The incorporation of G F P as a reporter enabled intracellular ScFv expression to be readily evaluated. A C terminal specific and a central domain specific anti-Nef ScFvs were studied for their capability for inhibition of the C D 4 and M H C - 1 downregulation effects induced by Nef. Neither one blocked the C D 4 and M H C - 1 d o w n modulation induced by Nef when co-expressed in P4.2 cells. Modification of these ScFvs w i t h N-terminal Nef myristoylation signal sequence fusion reduced the level of Nef expression significantly, probably due to more efficient binding and the subsequent sequestration of Nef. However, the myristoylation modification of these ScFvs d i d not make them effective inhibitors of C D 4 or M H C - 1  89  downregulation induced by Nef. It is concluded therefore that the binding interaction between ScFv w i t h N e f and the subsequent sequestration may not be sufficient to interfere w i t h its intracellular function. A ScFv or other intracellular ligands that could recognize and block epitopes i n N e f that are important for these functions w o u l d be required to have these specific effects.  90  CHAPTER 4 H C K S H 3 DOMAIN-DEPENDENT A B R O G A T I O N OF NEF-INDUCED CLASS I M H C DOWNREGULATION 4.1 Introduction  A s stated i n Chapter 3, while current anti-HIV therapy is successful i n keeping viral replication suppressed, it fails to eradicate the viral infection because of its inability to eliminate the reservoir of infected cell (Baiter, 1997). Therefore, therapeutic strategies toward the goal of eliminating the remaining H I V infected cells, i n combination w i t h current anti-HIV therapies that suppress viral replication, are desirable in the treatment of H I V infection. In this regards, Nef may be a potential therapeutic target for H I V infection, because of its role i n immune escape of H I V infected cells by downregulation of antigen presentation M H C - 1 molecules on cell surface (Collins and Baltimore, 1999). A m o n g the many detrimental effects of Nef on host cells, the M H C - 1 downregulation effect deserves special attention. Nef is the only HIV-1 viral protein that has been found to downregulate M H C - 1 in primary CD4+ T cells (Collins and Baltimore, 1999). This is i n contrast w i t h its role i n downregulation of C D 4 receptors w h i c h overlaps w i t h Env and V p u . Furthermore, the determinants of HIV-1 Nef w h i c h is responsible for M H C - 1 downregulation are distinct from that of the C D 4 downregulation. A s reviewed i n Chapter 1, these determinants include a conserved a-helix near the N-terminus of the protein, an acidic stretch i n the proximal region of HIV-1 Nef, EEEE65, and the polyproline motif forming the SH3-binding surface, PXXP78 (Greenberg et al, 1998b;  91  Mangasarian et al, 1999). The mechanism of downregulation of M H C - 1 induced by Nef remains to be elucidated; the LL165 dileucine motif w h i c h mediates the interaction of HIV-1 Nef w i t h adaptor complexes and is required for accelerated C D 4 endocytosis, is dispensable for M H C - 1 downregulation (Mangasarian et ah, 1999). Nef-induced M H C - 1 modulation was shown to protect HIV-1 infected cells against lysis by virus-specific CTLs in vitro (Collins et al., 1998). However, unless H L A - C or - E is expressed, downregulating M H C - 1 normally renders target cells more sensitive to destruction by N K cells (Brutkiewicz and Welsh, 1995; Colonna et al., 1993). Since these molecules are required for the negative control of N K mediated cytotoxicity. In this regards, Nef was shown to selectively downregulate M H C - 1 H L A - A , and H L A - B molecules, but not H L A - C or H L A - E (Collins and Baltimore, 1999; Le Gall et al, 1998). This selective downregulation of H L A - A and - B , but not - C , induced by Nef might therefore permit infected cells to display limited recognition for C T L s without being exposed to the attacks of N K cells.  In Chapter 3, the effect of Nef-specific single-chain antibody expression i n an attempt to block the C D 4 and M H C - 1 downregulation induced by Nef was examined. It was shown that the anti-Nef single-chain antibodies constructed retained the antigen-specificity of their corresponding parent antibodies. However, these ScFvs failed to inhibit the C D 4 and M H C - 1 downregulation induced by Nef. It is therefore concluded that the binding interaction and the following sequestration of Nef is not sufficient for blocking Nef functions, and an intracellular ligand that  92  could recognize the Nef determinants directly implicated in these receptor modulation effects are required to have maximum inhibition effect. A s a natural extension of the Chapter 3, the thesis research i n this chapter examines the possibility of inhibition of Nef functions by a dominant-negative H c k , a Src family of tyrosine kinases devoid of the kinase domain.  Several Src family tyrosine kinase are able to bind the polyproline (PXXP78) motif in N e f by their corresponding SH3 domains, with the H c k SH3 domain being able to bind N e f w i t h the highest affinity yet reported for an SH3-polyproline tract interaction ( K d = 0.25 uM) (Lee et al., 1995; Saksela et al., 1995). Taking advantage of this property, a dominant-negative H c k mutant, D N - H c k , consisting of the H c k amino-terminal domain, as well as the SH3 and SH2 domains was expressed i n H I V - l producing cells (Tokunaga et al., 1998). Interestingly, viral particles assembled i n the presence of D N - H c k demonstrated reduced infectivity suggesting that D N - H c k might be inhibiting an unknown Src family kinase activated by Nef at an early post-viral entry stage (Tokunaga et al., 1998).  In this thesis, it is hypothesized that D N - H c k might also be capable of interfering w i t h Nef-mediated effects through direct interaction w i t h Nef and hence blocking the downstream events induced by Nef. It is further hypothesized that the Nef-mediated effect that D N - H c k interferes with, is very likely the M H C - 1 downregulation effect, since the SH3-binding surface in Nef was implicated i n this effect as determined by mutation studies. In keeping w i t h this notion, this research revealed that D N - H c k prevented Nef-induced class I M H C downregulation, and this effect was dependent on the presence of a functional  93  H c k SH3 domain. Inhibition of the Nef-mediated effect demonstrated i n this work is of potential therapeutic importance, given that an intact Nef SH3-domain binding site is not only required for the M H C - 1 downregulation, it also has a number of other detrimental effects attributed to Nef during HIV-1 infection (Arold et al, 1997; Briggs et al, 1997; Collette et al, 1996; Goldsmith et al, 1995; Greenberg et al, 1998b; Iafrate et al, 1997; Mangasarian et al, 1999; Moarefi et al, 1997) .  4.2 Results 4.2.1 Dominant-negative  Hck binds Nef through its SH3 domain  Expression of the dominant-negative H c k (DN-Hck), its SH3 and SH2 mutants, as w e l l as a C r k l l control vector were evaluated i n transient transfection of P4.2 cells (Figure 23) (Tokunaga et al, 1998). The cells overexpressing these proteins were lysed and the cell lysates were examined for their ability to bind Nef crosslinked w i t h Sepharose beads in immuno-precipitation assays.  In addition to the w i l d type D N - H c k construct, two D N - H c k mutants were also used. The amino acid, Trp93, which was shown at the binding interface between N e f PP-II helix and F y n (R96I) SH3 i n the crystal structure of Nef-SH3 (corresponding to T r p l l 9 of SH3 described i n Literature Reviews) (Lee et al, 1996), was substituted with Phe i n DN-Hck-W93F construct (Tokunaga et al, 1998) . A r g l 5 1 , which is essential for the function of SH2, was substituted w i t h Ser in DN-Hck-R151S construct (Tokunaga et al, 1998). C r k l l adaptor protein, w h i c h consists mostly of the SH2 and SH3 domains, was used as a control for D N - H c k (Tokunaga et al, 1998).  94  A. —  SH3  H  SH2  B  SH3  H  SH2  SH3  H  SH2  —  Kinase domain  h  Wild-type Hck  pCAGGS-DN-Hck  pCAGGS-DN-Hck-R151S pCAGGS-DN-Hck-W93F  F i g u r e 2 3 S t r u c t u r e s of the w i l d - t y p e H c k ( A ) , the d o m i n a n t - n e g a t i v e m u t a n t f o r m of H c k ( B ) , a n d the two D N - H c k m u t a n t s ( C ) . ( R e p r o d u c e d f r o m T o k u n a g a et a l . , 1 9 9 8 )  4&  ^  C°  <?  N  jfr  2 •CrkII(32 kD) •DN-Hck (24.6 kD) .GFP (27 kD) . C r k l l (32 kD) •DN-Hck (24.6 kD)  Figure 24 A functional Hck SH3 domain is required for efficient binding of Nef to DN-Hck. A, Expression of DN-Hck in the P4.2 cells. Cells were electroporated with 15 ug of PCAGGS vector (control lane), pCAGGS-DN-Hck, pCAGGS-DN-HckW93F, pCAGGS-DN-Hck-R151S, or pCAGGS-Crkll, along with 4 ug of the pDEFGFP reporter plasmid. Cell lysates were collected 20 h late and analyzed by immunoblotting with rabbit anti-Hck antibodies orwith a monocloncal antibody against Crkll. The blot was stripped and reprobed with anti-GFP antibody. B, Immunoprecipitation of DN-Hck by recombinant Nef protein. P4.2 cell lysates were pre-cleared by GST cross-linked to Sepharose beads, then the Nef-GST fusion protein cross-linked beads were used to immunoprecipitate DN-Hck, its mutants and the control Crkll. The precipitated products were analyzed by immunoblot with anti-Hck antibody and the anti-Crk antibodies.SDS-PAGE was performed in 12.5% polyacrylamide gels under reducing conditions.  96  D N - H c k , its mutants and the C r k l l control were expressed at comparable levels in P4.2 cells (Figure 24 Panel A). Only D N - H c k and its SH2 mutant, D N - H c k R151S, were efficiently immunoprecipitated by Nef-GST cross-linked on Sepharose beads (Figure 24 Panel B). The binding interaction between D N - H c k w i t h N e f is specific, since there was no binding between N e f and C r k U , an adaptor protein w i t h SH2 and SH3 domains. The binding between D N - H c k R151S w i t h Nef was not affected by the A r g l 5 1 to Ser mutation, indicating that the SH2 domain is not directly involved in binding to Nef. However, the SH3 domain mutant-DN-Hck-W93F was not efficiently immunoprecipitated by Nef cross-linked on Sepharose beads, indicating that W93 residue is critical for the efficient binding interaction between these two molecules and that the H c k SH3 domain is responsible for the binding interaction with Nef.  To study the effects of D N - H c k and the two point mutants of this molecule on class I M H C expression i n Nef expressing cells, P4.2 cells were transfected w i t h Nef alone, or Nef plus each of the D N - H c k mutants (a representative experiment is shown i n Figure 25). Equivalent expression levels of all three D N - H c k molecules were obtained i n the transient expression experiments, and although expression of the D N - H c k molecules tended to be associated w i t h a modest reduction i n the levels of Nef expression (not seen w i t h the C r k l l control transfection), Nef (and G F P control) expression levels were similar i n the presence of vectors expressing D N - H c k , DN-Hck-W93F, and DN-Hck-R151S (Figure 25).  97  4? <c> S> f_cF ^  4^ t  cX  v  #^ cX  v  <^ CX  V  cX  ^  i "Crkll (32 kD) DN-Hck (24.6 kD) Nef (27 kD) GFP (27 kD)  Figure 25 Co-expression of DN-Hck mutant proteins and Nef in P4.2 cells. Cells were electroporated with 15 ug of AS and pCAGGS vector (control lane), 15 ug Nef vector together with pCAGGS (Nef lane), or Nef vector with HckN pCAGGSDN-Hck (Nef+HckN lane), pCAGGS-DN-Hck-W93F (Nef+HckN-W93F lane), pCAGGS-DN-Hck-R151S (Nef+HckN-R151S lane), or pCAGGS-Crkll (Nef+Crkll lane), along with 4 ug of the pDEF-GFP reporter plasmid in each case. Cell lysates were collected 20h post-transfection and analyzed by immunoblotting with rabbit anti-Hck antibodies and a monoclonal antibody that recognized Crkll (top row). After stripping, the immunoblot was first probed with anti-Nef antibody (middle row), and after re-stripping, with the anti-GFP antibody (bottom row). SDS-PAGE was performed in 12.5% polyacrylamide gels under reducing conditions.  98  4.2.2 Differential rate of CD4 and MHC-1 downregulation induced by Nef  Nef is w e l l k n o w n to downregulate both C D 4 and M H C - 1 receptors (Piguet et al., 1999b); In keeping w i t h this, surface expression of C D 4 and M H C - 1 receptors were significantly downregulated within 20 hrs after electroporation of P4.2 cells w i t h the Nef-expressing vector (Figure 26 a and f). M H C - 1 downregulation was less profound than that of CD4, likely due i n part to the observed differences i n turnover rates of these two molecules i n different cell types (Collette, 1997; York and Rock, 1996), and also to the observation that not all class I M H C molecules are subject to downregulation by Nef (Cohen et al., 1999; Le G a l l et at., 1998).  4.2.3 DN-Hck inhibits class I MHC downregulation induced by Nef  Flow cytometry analysis of P4.2 cells at 24 hr post-cotransfection w i t h plasmids of D N - H c k and Nef demonstrated that D N - H c k completely blocked Nef-induced class I M H C downregulation (Figure 26 g and Figure 27a). DN-Hck-R151S was equally effective i n blocking this Nef^ffect (Figure 26i; Figure 27a). However, expression of D N - H c k - W 9 3 F , a mutant predicted to have impaired SH3 function, failed to interfere w i t h Nef-induced class I downregulation (Figure 26h, Figure 27a). Likewise, C r k l l expression had no appreciable effect on class I M H C expression levels (Figure 26j, Figure 27a). Transfection of P4.2 cells w i t h increasing concentrations of the vector expressing D N - H c k revealed a dosedependent inhibition of Nef-induced class I downregulation (Figure 29). To control for the possibility that D N - H c k expression might itself be able to alter class I M H C cell surface expression, D N - H c k , DN-Hck-W93F, Hck-R151S, and C r k l l were transfected into P4.2 cells i n the absence of Nef, and M H C class I (and  99  CD4) levels were monitored by flow cytometry. N o significant change i n the cell surface expression of either molecule was observed as compared w i t h controls (Figure 28). Taken together, these results suggested that an interaction between the SH3 domain of D N - H c k and the SH3-binding surface of Nef might be the mechanism responsible for preventing the down-modulation of class I M H C downregulation caused by this viral protein.  These results demonstrate that D N - H c k , upon binding to the SH3-binding site of Nef, blocks the downstream interaction of Nef w i t h cellular proteins responsible for recruiting and targeting M H C - 1 molecules towards degradation pathway. The region on D N - H c k that is required for this action is the SH3 domain of the D N - H c k , since the SH2 domain mutant d i d not affect this blocking interaction.  4.2.4 DN-Hck partially affects CD4 receptor downregulation by Nef  The C D 4 receptor downregulation by Nef was partially blocked by coexpressing D N - H c k (Figure 26b and Figure 27a). The same effect was observed in cells co-expressing Nef and the SH2 mutant-DN-Hck-R151S (Figure 26d and Figure 27a). The SH3 mutant, DN-Hck-W93F, as well as the control vector expressing C r k l l failed to affect the C D 4 receptor downregulation i n the presence of Nef (Figure 26c, 26e and Figure 27a).  It is w e l l established that distinct determinants on Nef are used for C D 4 and M H C - 1 downregulation (Piguet et al., 1999b). Except for the amino-terminal myristoylation signal sequence of Nef, which is required for the membrane localization and critical for Nef to interact with both CD4 and M H C - 1 molecules,  100  Figure 26 The dominant-negative Hck blocks the CD4 and MHC-1 cell surface molecule downregulation effect induced by Nef. Surface C D 4 (a-e) and M H C - 1 receptor (f-j) levels i n P4.2 cells after transient transfection of Nef w i t h control vector (a and f), Nef w i t h D N - H c k (b and g), Nef w i t h D N - H c k - W 9 3 F (c and h), Nef with DN-Hck-R151S (d and i), as well as Nef and C r k l l (e and j) (open histograms). Control vector transfected cells: filled histograms.  101  102  Nef+Crkll Nef+DN-Hck-R151S Nef+DN-Hck-W93F Nef+DN-Hck Nef Control  Geometric mean values of MHC-1 fluorescence  Figure 2 7 S u m m a r y of C D 4 a n d M H C - 1 receptors down regulation-blocking effect by dominant negative Hck. T h e x a x e s represent geometric m e a n v a l u e s of M H C - 1 (a) a n d C D 4 (b) fluorescence from cells transiently transfected with M o c k , Nef, Nef a n d D N - H c k , Nef a n d D N - H c k - W 9 3 F , Nef a n d D N - H c k - R 1 5 1 S , a s well a s Nef and Crkll. Error bars represent the standard deviation from three independent experiments.  1 03  other motifs required for C D 4 downregulation are dispensable for the M H C - 1 downregulation, and vice versa (Piguet et al., 1999b). Furthermore, the SH3binding surface PXXP78 on Nef which binds with H c k was implicated only i n M H C - 1 downregulation, but not i n C D 4 downregulation. The reason for the partial blocking effect of D N - H c k on Nef-mediated C D 4 downregulation can be explained by the steric hindrance effect of D N - H c k when it binds the SH3binding surface i n Nef, rather than the direct blocking of Nef interaction w i t h downstream cellular proteins or Nef binding to CD4. The fact that both w i l d type and R151S mutants of D N - H c k , which bind with Nef, could partially block the C D 4 receptor modulation induced by Nef, but neither the SH3 mutant, D N - H c k W93F, nor the C r k l l control which do not bind w i t h Nef, interfere w i t h the C D 4 receptor downregulation i n the presence of Nef, supports the notion that binding of D N - H c k w i t h Nef on the SH3-binding surface is required for this steric hindrance effect.  4.2.5 DN-Hck does not modulate CD4 or MHC-1 receptor expression by itself  D N - H c k may also interact with cellular proteins directly and upregulate M H C - 1 and C D 4 receptors by activation of cellular signaling pathways. To rule out this possibility, P4.2 cells were transfected with D N - H c k alone and surface expression of receptors were evaluated by flow cytometry. N o significant cell surface C D 4 or M H C - 1 molecule modulation was observed (Figure 28a and 28e). Similarly, D N - H c k - W 9 3 F , Hck-R151S, and the control C r k l l do not have significant effect on cell surface expression of C D 4 and M H C - 1 molecules (Figure 28).  104  This indicates that the effect of the D N - H c k on C D 4 and M H C - 1 receptor downregulation by N e f is mainly through the direct interaction between D N H c k and Nef. The D N - H c k , upon binding w i t h the SH3-binding surface of Nef through its SH3 domain, effectively blocks the interaction of N e f w i t h the downstream cellular protein(s) responsible for M H C - 1 downregulation.  4.2.6 Dose response of dominant-negative Hck in Nef expressing cells  Electroporation of P4.2 cells with increasing amounts of plasmid expressing D N H c k demonstrated a saturable dose-dependent increase of M H C - 1 receptor i n the presence of Nef (Figure 29, upper panel). This further supports that the binding interaction, most likely between the SH3 domain of D N - H c k and the SH3-binding surface in Nef, plays a central role in the prevention of M H C - 1 receptor downregulation by Nef. There is also a slight dose-dependent increase of C D 4 receptors on P4.2 cell when D N - H c k was co-expressed w i t h Nef (Figure 29, lower panel). The magnitude of this increase is much smaller than that of the M H C - 1 . The mechanism of the latter effect is different from that of the M H C - 1 modulation and most likely because of inefficient C D 4 downregulation due to the increasing amount of Nef-complexed w i t h D N - H c k .  4.3 Discussion  The main hypothesis i n this thesis research is that during H I V infection class I M H C molecules are downregulated by Nef and without sufficient viral peptideclass I M H C antigen complexes on the cell surface, the HIV-infected cells escape the recognition and subsequent killing by CTL-the major host immunity against  105  Figure 28 The dominant-negative Hck alone does not modulate CD4 and M H C 1 cell surface molecule expression. Surface C D 4 (a-d) and M H C - 1 receptor (e-h) levels in P4.2 cells after transient transfection of D N - H c k (a and e), DN-Hck-W93F (b and f), DN-Hck-R151S (c and g), as well as C r k l l (d and h) (open histograms). Cells transfected by control vectors: closed histograms.  106  107  100  -i  90  H  Class 1 M H C  e o M UJ  l-i  o-  100 -i  X  <u  J  s-l 3  u  75  H  50  H  CD4  25 H  "T" o  o  Dominant-negative Hck vector (pCAGGS -DN-Hck, fig)  Figure 29 The dominant-negative Hck blocks the MHC-1 cell surface molecule downregulation induced by Nef in a dose-dependent manner. The geometric mean of MHC-1 (a) and CD4 (b) fluorescence in cells transiently transfected with Nef (15 ug) and increasing doses of vector expressing DN-Hck (2 to 30 ug) was shown on the ordinate, where 100% values correspond to the fluorescence level of cells transfected with the control vectors (pCMV-AS and pCAGGS). Error bars represent the standard deviation from three independent experiments.  108  HIV-1 infection (Collins and Baltimore, 1999). It is conceivable that by correcting this problem, the virus infected cells which could not be reached by conventional H I V reverse transcriptase and protease inhibitors could be eliminated. Nef may therefore represent a new target for therapeutic intervention i n individuals infected w i t h H I V - 1 .  The studies summarized i n this chapter were initiated because of the concerns that i n chapter 3, the single-chain antibodies targeting Nef failed to prevent C D 4 and M H C - 1 downregulation due to their 'non-essential' epitope binding characteristics, since none of the parent antibodies from the corresponding ScFvs bind to the determinants that are responsible for the C D 4 or M H C - 1 downregulation induced by Nef. In addition, it has been demonstrated previously that the targeting epitope of an intracellular antibody, rather than its binding activity, plays a pivotal role in disrupting the function of that viral protein (Rondon and Marasco, 1997).  The rationale for using a dominant-negative H c k to block the M H C - 1 downregulation induced by Nef is that the H c k SH3 domain was k n o w n to b i n d the polyproline (PXXP78) motif i n Nef w i t h the highest affinity ever reported for an SH3-polyproline tract interaction ( K d = 0.25 | i M ) . Furthermore, the Nef SH3 domain-binding PXXP78 motif is required for the M H C - 1 downregulation effect.  The results demonstrated that the dominant-negative H c k mutant, D N - H c k , is able to prevent Nef-induced M H C - 1 downregulation. The effect is H c k SH3 domain dependent as suggested by the inability of the D N - H c k - W 9 3 F mutant to  109  inhibit this effect. A s W93 is a key residue within the SH3 domain binding surface (Lee et al., 1995), it suggested that D N - H c k interferes w i t h Nef function by directly binding to the P X X P site of this molecule. The critical importance of W93 in the N e f - D N - H c k interaction was confirmed by an in vitro 'pull-down' experiment (Figure 24). This thesis also suggests that D N - H c k prevents Nefinduced class I M H C down-modulation by blocking the interaction between N e f and an as-yet unidentified SH3-containing cellular protein that is able to couple Nef to the M H C - 1 molecule. U p o n binding with Nef, this cellular protein might recruit class I M H C molecules via an interaction w i t h their cytoplasmic tyrosinebased sorting motifs, which in turn routes these molecules towards an intracellular degradation pathway (Greenberg et al, 1998b; Le G a l l et al, 1998; Mangasarian et ah, 1999).  A s the SH3-binding sites i n Nef are dispensable for C D 4 downregulation (Greenberg et al, 1998b; Mangasarian et al, 1999), the modest inhibition of Nefinduced C D 4 downregulation observed may have been due to either a weak steric hindrance effect, or possibly an allosteric effect subsequent to D N - H c k binding to the Nef SH3 domain binding sites. Since it has been established that distinct determinants on Nef are used to bring about C D 4 and M H C - 1 downregulation (Piguet et al, 1999b). W i t h the exception of the amino-terminal myristoylation sequence required for membrane localization and w h i c h is therefore critical for the interaction of Nef with both C D 4 and class I M H C molecules, the specific motifs required for C D 4 downregulation are dispensable for M H C - 1 molecule downregulation, and vice versa (Piguet et al, 1999b). Indeed, site-directed mutagenesis of the SH3-binding P X X P site of Nef has been shown  110  to inhibit Nef-induced class I M H C downregulation, but not C D 4 downregulation (Piguet et ah, 1999b). However, i n contrast to experiments that rely on introducing mutations into the sequence of Nef, the direct binding of the D N - H c k protein to Nef may also be capable of interfering, albeit inefficiently, w i t h Nef and C D 4 interactions. This may provide an explanation for the relatively modest effect of D N - H c k on Nef-induced C D 4 downregulation observed i n this thesis.  In conclusion, the research i n this chapter demonstrated that a dominantnegative form of H c k protein-tyrosine kinase was able to block Nef-induced downregulation of class I M H C surface expression in human cells. This effect required a functional SH3 domain, as it was not evident i n cells that express D N Hck-W93F, an SH3 domain mutation that results in diminished binding affinity for Nef. The results also suggest that this Nef-mediated effect requires an interaction between Nef and an as yet unidentified polyproline site-binding molecule. The SH3-region of Nef therefore represents a target for therapeutic intervention i n individuals infected with HIV-1.  Ill  CHAPTER 5 DISCUSSION  5.1 Summary of results  The objective of the studies presented i n this thesis was to examine the possibility of preventing Nef-induced immune dysfunction by intracellular expression of protein ligands. T w o important markers of immune dysfunction induced by Nef were studied, i.e. C D 4 and M H C - 1 expression. Expression of single-chain antibodies (ScFvs) against Nef were evaluated i n blocking C D 4 and M H C - 1 molecule downregulation induced by Nef. This was then extended to studies evaluating the effect of expressing dominant-negative H c k in interfering w i t h these Nef-mediated receptor downregulation effects.  In chapter 3, the effect of Nef-specific single-chain antibody expression i n an attempt to block the C D 4 and M H C - 1 downregulation induced by Nef was examined. It was shown that the anti-Nef single-chain antibodies constructed retained the antigen-specificity of their corresponding parent antibodies. However, these ScFvs failed to inhibit the C D 4 and M H C - 1 downregulation induced by Nef. It is therefore concluded that the binding interaction and the following sequestration of N e f is not sufficient for blocking Nef functions, and an intracellular ligand that could recognize the determinants i n Nef that is directly implicated i n these receptor modulation effects may be required to have the maximum inhibition effect.  112  In chapter 4, the possibility of inhibition of Nef functions by a dominant-negative H c k (DN-Hck), a Src tyrosine kinase devoid of the kinase domain, was examined. In vitro studies demonstrated that D N - H c k is able to prevent Nefinduced M H C - 1 downregulation. This effect is H c k SH3 domain-dependent as suggested by the inability of a DN-Hck-W93F mutant to inhibit this effect. A s W93 is a key residue within the SH3 domain binding surface (Lee et al, 1995), it suggested that D N - H c k was interfering w i t h Nef function by directly binding to the P X X P site of this molecule. The critical importance of W93 i n the N e f - D N - H c k binding interaction was confirmed by an in vitro 'pull-down' immunoprecipitation experiment (Figure 24).  5.2 Discussion and conclusion  Advances i n H I V research have made it possible to halt viral replication indefinitely, something inconceivable just several years ago (Bartlett and Moore, 1998). This is achieved by 'cocktail-therapy', i n which the patients have to take several drugs twice or more a day according to strict guidelines. A l l of the approved anti-HIV drugs are designed to block viral replication within cells by inhibiting either reverse transcriptase or the H I V protease, enzymes required for reverse transcription of viral R N A upon entry or viral protein post-translational processing during viral maturation, respectively.  Nevertheless, both scientific understanding and treatment remains far from perfect. None of the current therapies can eradicate the virus from patients once infected w i t h H I V and it is still unknown whether the impressive therapeutic responses from the 'cocktail-therapy' can be sustained.  113  A major new challenge for H I V therapy is finding ways to eliminate H I V from infected resting CD4+ T cells, which people used to believe that they d i d not actively produce viral particles but harbor the proviral D N A for doing so i n the future. To develop such therapies, new drug targets and alternative treatments such as gene therapy are under extensive research for their potential role i n the eradication of viruses from the infected resting cells (Bartlett and Moore, 1998; Rondon and Marasco, 1997).  A potential new drug target is Nef, a H I V viral protein w h i c h is implicated i n virus immune escape by protecting infected cells from recognition of cytotoxic T lymphocytes (Collins and Baltimore, 1999). A m o n g many other detrimental effects upon host cells, Nef is capable of downregulating cell surface C D 4 and M H C - 1 receptors w h i c h occur through distinctive determinants i n Nef (reviewed in (Piguet et al., 1999b)). The later activity deserves special attention, because it is linked to Nef's role i n protecting HIV-1 infected cells against lysis by virusspecific cytotoxic lymphocytes (Collins and Baltimore, 1999), w h i c h is the major host defence during H I V infection.  In this thesis, several anti-Nef single chain antibodies were first constructed from the variable regions of monoclonal antibodies that recognize the C-terminus and a central domain (Nef83-88) i n Nef (Figure 16 and Figure 20). A l l retained the binding activity of their corresponding parental monoclonal antibodies w h e n expressed intracellularly as demonstrated by 'pull-down' assays using recombinant Nef protein (Figure 18).  114  Two ScFv clones targeting the C-terminus and the central domain of Nef were studied for their potential inhibition of Nef-mediated effects. Neither had any significant effect on C D 4 and M H C - 1 downregulation induced by Nef. In comparison, transient transfection of P4.2 cells w i t h the corresponding N terminus myristoylation-modified ScFv vectors showed significant reductions i n the levels of Nef expression (Figure 21), w h i c h indicated that the addition of the myristoylation signal sequence to the ScFv clones made them more efficient i n reducing Nef expression level, possibly due to more efficient intracellular interaction w i t h their target protein. However, regardless of the N-terminal myristoylation status, none of the expressed ScFv was able to prevent C D 4 or M H C - 1 downregulation induced by Nef (Figure 22). This indicated that the intracellular binding of ScFv w i t h Nef and the resulting Nef sequestration may not be sufficient to prevent the receptor downregulation events induced by Nef. The expression of molecules capable of binding to epitopes i n Nef that are implicated specifically i n receptor modulation may be required for this effect. Others (Rondon and Marasco, 1997), have demonstrated that the affinity w i t h w h i c h a ScFv binds to the activation domain of HIV-1 Rev maybe significantly lower than if a different ScFv binds to a nonactivation region. However, the first ScFv demonstrated more potent activity in inhibiting virus production i n human T cell lines and P B M C s than did the later. Their results indicate that binding affinities of an ScFv for a target viral protein can not be used to predict its intracellular activity, and that the targeting epitope of the ScFv might play a more pivotal role i n disrupting the function of that viral protein (Rondon and Marasco, 1997).  115  A H c k mutant w i t h SH3 domain was then examined as a potential candidate for preventing^MHC-1 downregulation. Since the H c k SH3 domain binds Nef w i t h the highest affinity k n o w n for an SH3-mediated interaction ( K d = 0.25 fiM) (Lee et al, 1995). In addition, the SH3-binding motif (PXXP78) i n Nef is also one of the major determinants i n downregulation of M H C - 1 (Greenberg et al, 1998b). The implication of intracellular expression of dominant-negative H c k was therefore studied for receptor modulation effect in Nef-expressing P4.2 cells.  This research demonstrated that D N - H c k prevents Nef-induced M H C - 1 downregulation. This likely occurs via an interaction between the SH3 domain of D N - H c k and the P X X P motif i n Nef. The SH3-dependence of this effect was suggested by the inability of DN-Hck-W93F to inhibit this effect. A s W93 is a key residue forming the binding surface of H c k SH3 Domain (Lee et al, 1995), it indicated that D N - H c k was interfering w i t h Nef function by direct binding to the P X X P motif of Nef. The critical role of W93 i n the N e f - D N - H c k binding interaction was confirmed by the in vitro 'pull-down' immunoprecipitation experiment (Figure 24).  Several models have been proposed for the mechanism of Nef-induced class I M H C downregulation (Greenberg et al, 1998b; Le Gall et al, 1998; Mangasarian et al, 1999). It has been proposed that Nef recruits clathrin-adaptor protein complexes, w h i c h i n turn associate w i t h the cytoplasmic domain tyrosine-based sorting motifs within H L A - A and H L A - B molecules that have been shown essential for the Nef effect (Cohen et al, 1999; Greenberg et al, 1998b; Le G a l l et  11,6  al, 1998; Mangasarian et al, 1999). Alternatively, Nef could act as a connector between the class I M H C cytoplasmic domains and an adaptor protein complex (Greenberg et al, 1998b; Le Gall et al, 1998; Mangasarian et al, 1999). However, the adaptor protein complex and Nef have not been demonstrated to b i n d to class I M H C directly (Greenberg et al, 1998b; Le G a l l et al, 1998; Mangasarian et al, 1999). The involvement of a Src family kinase, or an SH3-containing protein lacking protein-tyrosine kinase activity in Nef-induced class I downregulation has also been proposed (Greenberg et al, 1998b; Le Gall et al, 1998; Mangasarian et al, 1999).  The results of this thesis strongly support a model that D N - H c k inhibits Nefinduced class I downregulation by preventing an interaction between Nef w i t h an as-yet unidentified SH3-containing cellular protein that couples N e f to the M H C molecule (Figure 30). U p o n binding with Nef, this cellular protein might recruit class I M H C molecules via an interaction w i t h their cytoplasmic tyrosinebased sorting motifs thus routing these molecules towards the intracellular degradation pathway (Greenberg et al, 1998b; Le Gall et al, 1998; Mangasarian et al, 1999) (Figure 30).  A s the SH3-binding sites i n Nef appear to be dispensable for C D 4 downregulation (Greenberg et al, 1998b; Mangasarian et al, 1999), the modest inhibition of Nef-induced C D 4 downregulation observed i n this thesis research may have been due to either a weak steric hindrance effect, or an allosteric effect, resulting from D N - H c k binding to the N e f SH3 domain-binding sites. It has been established that distinct determinants on Nef are used to bring about C D 4  117  and M H C - 1 downregulation (Piguet et al, 1999b). W i t h the exception of the amino-terminal myristoylation sequence that is required for membrane localization and is thus critical for interaction of Nef with both C D 4 and class I M H C molecules, the motifs required for C D 4 downregulation are dispensable for M H C molecule downregulation, and vice versa (Piguet et ah, 1999b). Thus, by site-directed mutagenesis of the SH3-binding P X X P site of Nef, this motif has been shown to inhibit Nef-induced class I M H C downregulation, but not C D 4 downregulation. However, i n contrast to such experiments that rely on mutating the sequence of Nef, the binding of the D N - H c k protein to Nef may be capable of interfering, albeit inefficiently, w i t h the interaction that occurs between N e f and C D 4 . This may provide an explanation for the modest effect of D N - H c k on Nef-induced C D 4 downregulation observed i n this thesis research.  The SH3-binding region of Nef represents a new target for therapeutic intervention i n individuals infected with HIV-1. Using a model system it has been shown that co-expression of Nef with a molecule, D N - H c k , that is capable of binding the Nef P X X P SH3-binding site, can reverse Nef-induced class I downregulation, despite the fact that Nef was likely being expressed at supraphysiological levels. While the effect of D N - H c k on class I expression i n cells infected w i t h HrV-1 needs to be further determined, it is conceivable that D N H c k w i l l also prevent M H C - 1 down-modulation i n these cells. In vivo such an effect w o u l d be predicted to increase the probability of infected cells being recognized and subsequently killed by C T L . In addition, since the Nef SH3binding site has been shown to play a role i n other viral activities, such as regulation of infectivity, cell activation and abnormalities of cell signal  118  Figure 30 Model for the dominant-negative Hck inhibition of MHC-1 receptor downregulation induced by Nef. A, Nef downregulates cell surface MHC-1 molecules through interation with an unidentified SH3-containing cellular protein that couples Nef to the MHC molecule. Upon binding with Nef, this cellular protein might recruit class I MHC molecules via an interaction with their cytoplasmic tyrosine-based sorting motifs thus routing these molecules towards the intracellular degradation pathway. B, DN-Hck inhibits Nef-induced class I downregulation by preventing this interaction between Nef and that unknown cellular protein.  119  transduction pathways (Arold et al, 1997; Briggs et al, 1997; Collette et a/., 1996; Goldsmith et al, 1995; Iafrate et al, 1997; Moarefi et al, 1997), therapies directed against this Nef site could have additional benefits for the host.  There are several potential limitations i n this thesis research. First, due to limited resources, only m R N A from hybridomas raised against 'non-essential' regions of Nef were obtained. Therefore, ScFv constructed from the c D N A of these hybridomas were limited by their corresponding binding specificities. Ideally, ScFv could be generated from hybridomas producing antibodies targeting epitopes on N e f that are implicated i n C D 4 or M H C - 1 downregulation induced by Nef. Such ScFvs are more likely to have a suppressive effect on C D 4 or M H C 1 downregulation induced by Nef. Second, the P4.2 cell line transient transfection system used to evaluate potential intracellular anti-Nef reagents is an in vitro assay i n a research laboratory setting. The effect of D N - H c k i n preventing M H C 1 downregulation induced by Nef suggests, but does not directly prove, the potential clinical benefit using such a therapeutic strategy. O n l y after extensive in vitro and in vivo testing i n HIV-1 infection models, the therapeutic strategy suggested from this thesis research can be established.  It is worth to emphasize that cytotoxic T-lymphocyte (CTL) responses arise early after H I V or SIV infection and are important i n controlling viral replication throughout the course of infection (Borrow et al, 1994; K o u p and H o 1994; Yasutomi et al, 1993; Reimann et al, 1998; Ogg et al, 1998; Schmitz et al, 1999; Jin et al, 1999; Matano et al, 1998). A number of clinical and experimental observations have implicated virus-specific CTLs i n this process. CD8+  120  lymphocytes from infected individuals have been shown to inhibit HIV-1 replication i n vitro (Walker et al, 1986). Control of the surge of viral replication i n primary HIV-1 infection coincides w i t h the appearance of virus-specific C T L s (Koup et al., 1994). Potent virus-specific C T L responses have been observed i n infected individuals w i t h low viral loads and persistent, nonprogressive infections (Rinaldo et al, 1995; Ogg et al, 1998). In addition, the importance of C T L s i n controlling STY has been demonstrated by deleting this cell population using anti-CD8 monoclonal antibody in macaques infected w i t h SIV, w h i c h resulted i n a rapid and marked increase i n viremia that was again suppressed coincident w i t h the reappearance of SIV-specific CD8+ T cells (Jin et al, 1999; Schmitz et al, 1999). These results confirmed the crucial role of cell-mediated immunity i n controlling HIV-1 infection.  One future application from this thesis research w i l l be to develop anti-Nef therapy aimed at preventing the M H C - 1 downregulation effect induced by Nef during HIV-1 infection. This therapeutic strategy, once developed, could be used for assisting the host immune system to eliminate HIV-l-infected cells. It is hopeful that together w i t h the powerful "cocktail-therapy" that suppresses viral replication, H I V infection can one day be cured. But there is one potential limitation for the anti-Nef approach: it only works if there is H I V protein expression i n the infected cells. It w i l l not enhance viral antigen presentation by M H C - 1 , i n order for CTLs to better recognize and k i l l viral infected cells, i n a truly latent viral state; since i n such a state there is no viral protein expression. Current evidence supports that the dormant reservoir of H I V w h i c h is established early during primary infection (Chun et al, 1998), consists of latently  121  infected, resting CD4+ T cells carrying replication-competent H I V (Chun et al, 1999). This pool of infected cells can persist even i n individuals who are receiving highly active antiretroviral therapy ( H A A R T ) (Chun et al, 1997; C h u n g et al, 1997; Finzi et al.,1999; Zhang et al, 1999), and viral replication rapidly rebounds from this reservoir w i t h i n weeks of discontinuing H A A R T treatment (Chun et al, 1999). Clinical evidence has also shown that H I V continues to replicate at a l o w level even i n patients w i t h undetectable levels of virus in their blood (Ho, 1997). A l t h o u g h these studies d i d not demonstrate directly the viral protein expression status i n those latently infected cells, they indicated that there might not be a truly latent viral state, viral protein production i n those infected cell might be on all the time. The only limitating factor for the viral protein production are the drugs inhibiting the reverse transcriptase and protease of HIV-1. Even if the latent infection of H I V means complete silence of viral replication, anti-Nef therapy can still be effective against the re-activation of H I V i n the latently infected cells or prevent the infection from the latent state to replication competent.  It should be emphasized that there is still a long way to go before translating the results generated from this thesis into H I V therapy. In future studies, it w i l l be necessary to prove that the inhibition effect on the Nef-induced M H C - 1 downregulation affects the ability of the CTLs to kill virus infected cell i n an in vitro model of HIV-1 infection. This can be achieved by the method used for studying the protective effect of the expression of Nef i n infected cells against anti-HIV C T L recognition (Collins et al, 1998). Basically, primary CD4+ T cells are infected w i t h an H I V that can place a reporting marker, such as G F P or  122  placental alkaline phosphatase (PLAP) i n the infected cells. Then, C T L clones restricted to M H C class I H L A - A 2 antigens and the G a g epitope ( S L Y N T I A V L (SL9)) could be used (Collins et al, 1998). D N - H c k or other Nef P X X P ligands, such as ScFv binds to the P X X P domain i n Nef, can be introduced in cis within the H I V vector. This w i l l allow D N - H c k or other Nef-binding agents to be coexpressed intracellulary with Nef i n cells infected by this modified H I V vector. Alternatively, cells used for infection can also be pre-transfected w i t h vectors carrying the c D N A s of these ligands. The cytotoxic activity of these C T L s towards HIV-infected cells can be evaluated and the protective role of therapies targeting the SH3-binding surface of Nef can then be established in vitro.  Second, the in vitro benefit of inhibition of Nef-induced M H C - 1 downregulation has to be further tested in an H I V infection animal model. Ideally, this animal model can be SIV infection of chimpanzee or macaques, since they are the closest animal models to H I V infection i n human. The clinical benefit of such a therapeutic strategy can be monitored by measuring viral loads RT-PCR, or examination of infected cells from local l y m p h node or peripheral blood. Small molecules that can be selected by rational drug design according to the structure and conformation of Nef protein, or peptides that can be selected according to their b i n d i n g affinity to Nef protein, could be developed as drugs targeting this Nef SH3-binding domain. The advantage of small molecules is that they can be administered by oral and intravenous administration. It is expected that the combined H I V therapeutic scheme using drugs targeting the SH3-binding surface on Nef, together with inhibitors suppressing H I V replication, w i l l be  123  more effective i n controlling H I V infection and may eventually reach the goal of eradicating H I V infection.  One potential application suggested by this research is gene therapy. Gene therapy is a new form of molecular medicine that has gained special interest among A I D S researchers, since conventional therapies have shown limited success. Alteration of the host cell could potentially confer permanent suppression of viral replication after infection, or could provide protection against viral infection. Several gene therapy strategies for H I V infection are currently being studied, including immune reconstitution w h i c h involves ex vivo expansion of selected and sometimes genetically modified T cells, followed by their reinfusion into the infected patients (Roberts et al., 1994); nucleic acid-based therapeutic vaccines involve direct delivery of HIV-1 genes to mimic viral infection wherein the expression of viral proteins encoded by these nucleic acids elicits both cellular and humoral response (Wang et al., 1993); lastly, intracellular immunization transfers a therapeutic gene into target cells to render them resistant to viral infection, the resistant cells w i l l then limit the spread of the virus i n the patient (Baltimore, 1988).  Several research groups have successfully constructed and characterized ScFvs against different HIV-1 structural and regulatory proteins. ScFvs targeting H I V - 1 envelope protein gpl20 have been shown to inhibit the envelope protein processing i n E R and reduce infectivity of HIV-1 particles released by ScFv expressing cells (Marasco et al., 1993). Currently, a clinical trial is i n progress using conventional gene therapy vectors containing these ScFvs to transduce and  124  reinfuse autologous CD4+ T cells i n asymptomatic HIV-1 infected patients (Rondon and Marasco, 1997). This gene therapy approach may provide a therapeutic benefit for HIV-1 infected patients. ScFvs targeting other HIV-1 viral regulatory and structural proteins, such as Tat, Rev, reverse transcriptase, integrase, matrix and nucleocapsid, have also been generated. Each of them exhibited various degrees of inhibition of viral infection and cell protection (Duan et al, 1994a; D u a n et al, 1994b; Levin et al, 1997; Levy-Mintz et al, 1996; Maciejewski et al, 1995; Mhashilkar et al, 1995; Shaheen et al, 1996; W u et al, 1996). In summary, a variety of HIV-1 proteins are sensitive to neutralization b y intracellular antibodies, and H I V inhibition can be achieved at different stages of the viral life cycle. These intracellular antibodies may be useful for the gene therapy of HIV-1 infection.  The knowledge obtained from this thesis can be used to develop a novel gene therapy that targets H I V infected cells. Retroviral vectors pseudotyped w i t h C D 4 and different chemokine receptors can be used to carry genes encoding ligands targeting the P X X P region i n Nef and transduce the reservoir of HIV-infected cells. The entry of HIV-1 is mediated by interactions between the viral glycoprotein and a cellular receptor complex, which consists of C D 4 and one of the C C or C X C chemokine receptor family proteins; and it has been demonstrated that these viral receptors can be used to target HIV-infected cells (Endres et al, 1997). The ligands can be D N - H c k , single-chain antibody or peptide specific for the P X X P region i n Nef.  125  Basically, a retroviral vector coated with functional H I V viral receptors can be made b y co-transfection the viral packaging cells with plasmids encoding CD4, a chemokine receptor (CXCR4 or CCR5), and an envelope-deficient HIV-1 vector that encodes a protein ligand for the P X X P region i n HIV-1 Nef. These receptorpseudotyped virions can be examined for the ability to enter HIV-infected cells, as w e l l as the ability to interfere w i t h Nef functions intracellularly. Such vectors w i l l be useful to prevent M H C - 1 downregulation induced by Nef, and i n doing so, might let the host HIV-1 specific CTLs better recognize and remove the infected cell.  In conclusion, a strategy was first established to construct and evaluate Nefspecific single-chain antibodies; however, intracellular single-chain antibody expression against 'non-essential' regions on Nef d i d not prevent C D 4 or class I M H C downregulation induced by Nef. The dominant-negative H c k was then evaluatedq and it was demonstrated that the dominant-negative H c k expression inhibited M H C - 1 downregulation induced by Nef, and this effect is H c k SH3 domain dependent. A model was proposed that D N - H c k inhibits Nef-induced class I downregulation by preventing an interaction between Nef w i t h an u n k n o w n SH3-containing cellular protein that links Nef to the M H C molecule. 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