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Studies on HIV-1 Virion Infectivity Factor Studies on HIV-L Virion Infectivity Factor Feng Feng M.Sc. Infectious Diseases Laboratories Institute of Medical and Veterinary Science Adelaide South Australia School of Molecular & Biomedical Science University of Adelaide Adelaide South Australia ¡k ¡l.rl. {. t {. {. ¡1. ¡ß {. tk * *,1. tß * * * * {. A thesis submitted to the University of Adelaide infuffilment of the requirements far the degree of Doctor of, Philosophy October,2004 Contents VI Declaration of Originality.... .................. IX X Publications Related to this Study xll Chapter L. IntrOduCtiOn... .................. ... .... o. ........ 1 1.1 Historical Background of HMAIDS 1..2 Overview of Human Immunodeficiency Virus............ 4 1.2.1 Classification of HIV 4 1.2.2 }J.IV virion structure 5 1.2.3 Genetic organisation of HIV 5 6 1 .2.4 IF'IV replication CYcle. 1.2.4.t Viral attachment and viral fusion 7 1.2.4.2 Reverse transcription. .' . I 1.2.4.3 Viral DNA integration.. '... 9 I.2.4.4 Transcription and translation l0 1.2.4.5 Assembly of virus and viral budding. 1l 1.2.5 HIV gene expression. t2 1.2.5.1HIV proviral genome transcription - overview ... ' l2 I.2.5.2 Regulation of transcription' ' l3 1.2.6 I{IV encoded proteins and their functions. l5 I.2.6,1 The major structure proteins. 15 I.2.6.2 Regulatory proteinsiaccessary proteins. .' l8 1.2.t HIV-1 Virion infectivity factor (ViÐ'.. 24 1.2.7.1HIV-l Vif localisation and biological activity' 24 112.7 .2 Vif is required for efficient reverse transcription. .. 25 I.2.7.3 Vif interacts with viral proteins and RNA. 27 I.2.7.4 The effect of Vif is cell type dependent and involves cellular inhibitors of HIV replication. 29 1.2.7 .5 Host cellular factors that interact with Vif . 30 1.2.8 Pathogenesis of HIV-1 Infection. 34 1.2.8"1 Signs, symptoms of primary HIV-1 infection'.... 34 I.2.8.2 Aspects of virus-host interactions affecting pathogenesis...... 35 ll 1.2.8.3 Developments in viral pathogenicity 5t 1.3 IIMAIDS vaccine developments. 39 1.4 HIV/AIDS treatment..... 40 1.5 Summary.. ........................... 43 1.6 Hypotheses....... ...... 44 1.7 Specific Aims 45 Chapter 2. Materials and methods 46 46 2.1.1 Cells and cell culture. 46 2.1 .2 Bacterial culture 47 2.1.3 Yeast 4H109 and cell culture. 48 2.1 .4 Plasmid vectors. 48 2.1.5 Oligonucleotide sequences. 49 2.1.6 Antibodies. 51 2.I.7 Commonly used buffers and solutions'. 5t 2.2.r Investigation of Vif/Triad 3au or NVBP interactions in yeast....'. 54 2.2.2 Production of Triad 3au DNA Probe. 55 2.2.3 RNA extraction and Northern blot analysis..... 56 2.2.4 Pol)'rnerase chain reaction (PCR). 57 2.2.5 Determination of the full-length Triad 3au oDNA sequence ...... 58 2.2.6 DNA constructions. 59 2.2.7 Transient cell transfection 60 2.2.8 Western blotting 6l 2.2.9 Co-immunoprecipitation analyses. 62 2.2.I 0 Immunofluorescent staining. 63 2.2.11GST fusion protein pull-down assay. 64 2.2.12 Production of HIV NL4.3 stocks' 66 2.2.13 Single-cycle viral infectivity assay. 66 2.2.14 Viral titres (TCIDso) 67 2.2.I5 Extent of reverse transcription in newly infected target cells.... 68 2.2.16 Site-directed mutagenesis of HIV-1 Vif' 69 Chapter 3. Interaction between HIV-Vif and Tfiad 3aU... ... ... ..... t...... .. " " t t " " " "" o" 7l 7l lll 3.1.1 NVBP........... 11 3.1.2 Triad3au........ 72 3.2 Interaction in yeast between HIV-I Vif and Triad 3au or NVBP 75 3.3 Interaction between Triad 3au and HIV Vif in mammalian cells..................o...... ............ 77 3.4 Effect of Triad 3au on the cellular localisation of Vif.... 79 3.5 Triad 3au mRllA is expressed in both permissive and primary non-permissive cells. 8t 3.6 Identification of ZIN and preparation of the ZIN cI)NA. ... ... ... ... ... ... .. ... ... ... ... .... 83 Chapter 4. Interaction between HIV-1 Vif and ZIN' and Implications for Viral Replication...... 87 4.1 Introduction. ...... ............ 87 4.2 Z.,IN interacts with purified Yil in vitro... ... 88 4.3 ZIN interacts with HIV-I Vif in mammalian cells. 90 4.4 Co-localisation of Vif and ZIN in transfected cells 9l 4.5 Effect of ZIN overexpression on production and infectivity of HIV-I............. ...... .......... 94 4.5.1 TCID5g analysis of wild-type HIV produced in the presence or absence of co-transfected ZIN. .. 94 4.5.2 Single-cycle viral infectivity assay of wild type and Lvif HIY in the presence or absence of co-transfected ZIN. 95 4.5.3 Real time PCR analysis of viral DNA synthesis after infection....' 97 4.5.4 Discussion... 98 Chapter 5. Vif Site-Directed Mutagenesis 100 5.1 Introduction ............ 100 5.2 Experimental principle 101 lv 5.3 Result and discussion..........................................'. t02 'Work....... Chapter 6. General Discussion and Future 104 6.1 DiSCUSSiOn..........................'.............................. 104 6.2 FUtUre WOfk... ... ... ... ... ... ...... ... ... ... ... ...... ... ... ... ... I 14 Referenc€.... o................................................... ll8 V Abstract Virion Infectivity Factor (Vif) protein of human immunodeficiency virus type I (HIV-l) is essential for the productive viral infection of primary human CD4 T lymphocytes and macrophages. Recently, it has been reported that Vif overcomes the HlV-inhibitory effects of the cellular factor CEMI5/APOBEC3G, which has cytidine deaminase activity. Using the yeast two-hybrid system, our laboratory previously reported the identification of a Vif-interacting ring finger protein called Triad 3 (renamed Triad 3au in this study), ftom a human leukocyte oDNA library. The full-length cellular protein homologue of Triad 3 has been recently identified as the zinc finger protein inhibiting NFkB (ZIN). In this study, biological characteristics of Triad 3au/ZIN were investigated. Sequence analysis indicated that Triad 3au protein contains all 4 major ring-like motifs of ZIN. RNA was extracted from A3.01 cells and RT PCR was then performed using the ZIN gene specific primers. A single product of 1,464 bps was obtained and subsequently confirmed as the full-length coding region of ZIN by sequence analysis. GST fusion protein pull-down experiments confirmed that overexpressed ZIN binds to purified Yif in vitro suggesting a direct interaction between ZIN and Vif. Next, in studies of co- transfected human 293T cells, Triad 3ar.r/ZIN and Vif were shown to interact using co-immunoprecipitation and confocal microscopy demonstrated co-localisation of Triad 3au and Vif in cytoplasm and membrane while co-localisation of ZIN and Vif in nuclei. To test the biological relevance of the Vif-ZIN interaction, infectious HIV-I NL4.3 virus stocks were produced in 293T cells expressing Flag tagged ZIN or Flag tag VI alone from transfected plasmids. The virus stocks produced in the presence of exogenously expressed ZIN were less infectious in both single-cycle infectivity assay and end-point titration compared with virus produced in the absence of exogenous ZIN. Real Time PCR analyses showed that cells infected with HIV NL4.3 virus stocks produced in the presence of exogenously expressed ZIN, showed reduced levels of early viral DNA synthesis. This reduction in viral reverse transcription and the reduction in single-cycle viral infectivity were shown to be dependent on the presence of Vif in the virus producer cells. The possible mechanisms by which presence of ZIN in producer cells reduces HIV-1 reverse transcription and replication in target cells are discussed. vll Acknowledgments This work was supported by the International Postgraduate Research Scholarship (IpRS) and Adelaide University Scholarship. This research was in part supported by a grant from the Australia Commonwealth AIDS Research Grant Programme. Many thanks go to Dr. Peng Li and Prof. Chris Burrell for giving me the opportunity to pursue my Ph.D program in Australia and their invaluable guidance throughout the course of this Ph.D. Dr. Adam Davis, many thanks for your patience and time with my project and correcting my paper and thesis. To Dr. Julie Lake, thank you very much for your teaching at the early stages of my Ph.D study. To Dr. Jill Can, Dr. Raman Sharma and Dr. Nick Vandegraaff, thank you for your guidance. You were always available whenever I had trouble with my project over the past years. A great thank to Jennifer Clarke, Jane Arthur and Ghafer Sarvestani, for your technical guidance and useful discussions with my immunofluorescence experiments' A special thankyou to Adrian Purins and Kelly Cheney for your jokes, laughs, all questions raised during my laboratory talks and other supports. These are highly appreciated. To the entire HIV lab, both present (Carl Coolen, John Karlis, Satiya Wati, Sarah Martin, Branka Bauk) and past (Linda Mundy, Helen Hocking, Luke Dimasi, Robyn Taylor) for their helpful discussions and good friendships. Also Dr. Tuckweng Kok, thank you for your help with reviewing the figures and figure legends of my thesis' To my family and relatives, thank you all very much for your understanding and utmost support during my studies. vlll Abbreviations less than greater than OC degrees Celcius AIDS Acquired Immune Deficiency Syndrome bp base pair Ci curre dATP 2' -deoxyadenosine 5' -triphosphate dCTP 2' -deoxy cytidine 5' -triphosphate DDW double distilled water dGTP 2' -deoxynucleoside 5'-triphosphate DNA deoxyribonucleic acid dNTP 2'-deoxynucleoside 5' -triphosphate DTT dithiothreitol dTTP 2' -deoxythymidine 5' -triphosphate EDTA ethylene-diamine-tetra-acetic acid g(unit) gravity force HIV-1 Human Immunodeficiency Virus Type I hr hour(s) IN integrase Kb kilobase-pair LB Luria-Bertani medium trg microgram pM micromolar (micromoles per litre) mln minute(s) ml millilitre rnM millmolar (millimoles per litre) ng nanogram OD optical density nt nucleotide 32P radioactive phosphorous (mass number: 32) PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PIC preintegration complex RNA ribonucleic acid rpm revolutions per minute RT reverse transcriptase SDS sodium dodecyl sulphate S second(s) SSC standard saline citrate ssDNA Strong-stop DNA TCIDso 50% tissue culture infectious dose XI Publications Related to this Study l.
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