Investigation of the genetic and molecular basis of HIV-1 cellular tropism in vivo

X CHAPTER 1: GENERAL INTRODUCTION 1 1.1 HISTORICAL PERSPECTIVE 2 1.1.1 Classification and morphology of retroviruses 4 1.1.2 Origins andGlobal Diversity of HIV 6 1.2 THE HIV-1 VIRION 8 1.2.1 Genomic organisation of HIV-1 10 1.2.2 Structural proteins 12 Gag 12 Pol 14 Env 16 1.2.3 Regulatory proteins 21 Tat 21 Rev 22 1.2.4 Accessory Proteins 23 Nef 23 Vif. 24 Vpr 24 Vpu 25 1.3 THE HIV-1 LIFE CYCLE 26 1.3. 2 Reverse transcription 32 1.3. 3 Integration 33 1.3. 4 Transcription of viral DNA 35 1.3. 5 Assembly and Budding 36 1.4 PATHOGENESIS OF HIV-1 37 i 1.4. 1 Transmission of HIV-1 37 1.4. 2 Disease Progression 38 1.4. 3 Viral Dynamics and Immune Responses 39 1.4. 4 Antiviral Therapy 42 1.5 GENETIC DIVERSITY OF HIV-1 43 1.5. 1 Sources of mutation during HIV replication 44 1.5. 2 Viral turnover in HIV infected individuals 47 1.5. 3 Fixation rate of mutations in HIV 49 1.6 RECOMBINATION IN HIV-1 54 1.6. 1 Molecular mechanisms of retroviral recombination 55 1.6. 2 Recombination between global HIV-1 isolates 60 1.6. 3 intrasubtype recombination 63 1.6. 4 Recombination between closely related HIV-1 strains 64 1.7 MOLECULAR DETERMINANTS OF TROPISM 66 1.7. 1 CD4 66 1.7. 2 coreceptors 68 1.7. 3 Further factors involved in viral entry 74 1.7. 4 Envelope determinants of HIV-1 cellular tropism and coreceptor use 77 1.7. 5 CD4 and coreceptor independent interactions 80 1.8 COMPARTMENTALISATION OF HIV-1 83 1.9 HIV-1 AND THE BRAIN 84 1.9. 1 Neurological symptoms and pathology associated with HIV-1 infection 85 1.9. 2 Timing of HIV-1 infection of the brain 87 1.9. 3 Infected cell types in the brain 88 1.9. 4 HIV-1 induced damage in the CNS 89 1.9. 5 HIV-1 nucleotide sequences in the brain 90 1.9. 6 Pfienotype of HIV-1 in the brain 92 1.10 MOLECULAR EVOLUTION 97 1.10. 1 Distance Measures forNucleotide Sequences 97 1.10. 2 Molecular Phylogenetics 98 1.10. 3 Statistical Significance of Tree Topologies 100 1.11 AIMS 100 2: MATERIALS AND METHODS 103 2.1 AUTOPSY SAMPLES 104 2.2 BIOLOGICAL SAFETYWHEN WORKING WITH HIV 104 2.3 DNA EXTRACTION 105 2.4 QUANTITATION OF EXTRACTED DNA 106 2.5 POLYMERASE CHAIN REACTION 107 2.5. 1 General procedures 107 2.5. 2 Primer sequences 107 2.5. 3 PCR OF Pl7GAG, V1/V2, V3, VET AND EGFP 109 Reagents 109 Protocol 110 Thermal cycling conditions 110 2.5. 4 PCR of full-length eat' and luciferase genes 1 11 Reagents 112 Protocol 112 Thermal cycling conditions 112 2.6 AGAROSE GEL ELECTROPHORESIS 112 Reagents 112 Protocol 113 2.7 QUANTITATION OF HIV-1 PROVIRAL DNA 113 2.8 CLONING OF PCR PRODUCTS 113 2.8. 1 Phosphorylation of PCR products 114 2.8. 2 Ligation reaction 114 2.8. 3 Transformation 115 Reagents 115 Protocol 116 2.8. 4 Colony Screening 116 2.8. 5 Plasmid DNA Mini-preps 117 Reagents 117 Protocol 117 2.8. 6 Plasmid DNA Midi-preps 118 Protocol 118 2.9 RESTRICTION DIGESTS 119 2.9. 1 pme I digest 119 Reagents 119 Protocol 120 2.9. 2 Hind III andXmnI single and double digests: 120 Reagents 120 Protocol 120 2.10 OVERLAP EXTENSION PCR REACTIONS 121 2.11 PCR USING LIGATION REACTIONS AS TEMPLATE 121 2.12 EXTENSION REACTIONS 122 2.13 FINAL AMPLIFICATION 122 2.14 PCR PRODUCT PURIFICATION 123 2.15 DNA SEQUENCING 124 2.15. 1 Manual Sequencing 124 Reagents 124 Protocol 125 2.15. 2 Acrylamide Gel Electrophoresis 126 Reagents 126 Protocol. 126 2.15. 3 Automated Sequencing 127 Reagents 127 Protocol 127 2.16 SEQUENCE ANALYSIS 128 2.17 EUKARYOTIC CELL CULTURE 132 2.17. 1 Growth medium and Cell Lines 132 2.17. 2 Thawing Cells 132 2.17. 3 Passaging Cells 133 2.17. 4 Freezing cells 133 2.18 ELECTROPORATION OF 293 T CELLS 133 2.19 FLUORESCENT MICROSCOPY 134 2.20 LUCIFERASE ASSAY 134 2.21 NEF IMMUNOSTAINING 135 2.22 PSEUDOTYPE VIRUS PRODUCTION AND ANALYSIS 136 2.23 CALCIUM PHOSPHATE MEDIATED TRANSFECTION: 138 2.24 VIRAL INFECTIVITY ASSAYS 139 2.25 P24 IMMUNOCYTOCHEMISTRY 140 CHAPTER 3: RESULTS (I) 142 3.