VARIATIONS IN THE V3 CROWN OF HIV-1 ENVELOPE IMPACT AFFINITY FOR CCR5 AND AFFECT ENTRY AND REPLICATIVE FITNESS By MICHAEL ANDREW LOBRITZ Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisor: Eric J. Arts, Ph.D. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY August 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents Chapter 1: Introduction........................................................................................................................16 1.A. HIV and AIDS..............................................................................................17 1.B. Retroviruses: Structure, Organization, and Replication...............................20 1.B.1. HIV-1 Genome...............................................................................20 1.B.2. HIV-1 Particle................................................................................23 1.B.3. HIV-1 Replication..........................................................................26 1.B.3.a. Transmission and Replication Dynamics........................26 1.B.3.a. Host Cell Tropism and Replication.................................26 1.B.3.b. HIV-1 Envelope and Host Cell Entry.............................29 1.B.3.c. Reverse Transcription......................................................33 1.B.3.d. Nuclear Translocation and Integration............................36 1.B.3.e. Transcription and RNA Export.......................................39 1.B.3.f. Assembly, Budding and Maturation................................42 1.B.3.g. HIV-1 Accessory Proteins...............................................43 1.D. Antiretroviral Therapy...................................................................................44 1.E. HIV-1 Receptors and Implications of Tropism.............................................47 1.F. Interaction of HIV-1 Envelope and CCR5....................................................50 1.G. Inhibition of HIV-1 Entry.............................................................................55 1.G.1. Attachment Inhibitors....................................................................55 1.G.2. Fusion Inhibitors and Mechanisms of Resistance..........................57 1.G.3. Coreceptor Inhibitors and Mechansims of Resistance...................59 2 1.G.3.a. Chemokine Analogs........................................................59 1.G.3.b. Small Molecule Antagonists...........................................61 1.G.3.c. Models of Resistance to Coreceptor Inhibitors...............62 1.H. Implication of HIV-1 Entry Efficiency.........................................................68 1.I. Hypotheses......................................................................................................70 Chapter 2: Natural Variation in the V3 Crown of Human Immunodeficiency Virus.......72 Type 1 Affects Replicative Fitness and Entry Inhibitor Sensitivity 2.1. Preface............................................................................................................73 2.2. Abstract..........................................................................................................74 2.3. Introduction....................................................................................................75 2.4. Materials and Methods...................................................................................78 2.4.1. Cells and Viruses...........................................................................78 2.4.2. Western Detection of Viral Proteins..............................................80 2.4.3. Single-cycle Infection Assays........................................................80 2.4.4. Entry Inhibitor Sensitivity..............................................................81 2.4.5. Replicative Fitness from Competition Assays...............................82 2.5. Results............................................................................................................84 2.5.1. Natural Variation at HIV-1 V3 Positions 318 and 319..................84 2.5.2. Replicative Fitness of V3 Mutant Chimeric Viruses in PBMC.....94 2.5.3. Replication of V3 Mutant Chimeric Viruses in...........................106 Macrophage Cultures 2.5.4. Antiretroviral Susceptibility Conferred by V3 Polymorphisms...108 2.5.5. Comparing Replicative Fitness with Sensitivity to.......................116 3 Entry Inhibitors 2.6. Discussion....................................................................................................119 Chapter 3. Differential Sensitivity to Inhibition of Human Immunodeficiency.............128 Virus Type 1 to PSC-RANTES Involves Competitive CCR5 Binding 3.1. Preface..........................................................................................................129 3.2. Abstract........................................................................................................130 3.3. Introduction..................................................................................................131 3.4. Materials and Methods.................................................................................135 3.4.1. Reagents.......................................................................................135 3.4.2. Plasmids.......................................................................................135 3.4.3. Cells.............................................................................................135 3.4.4. Viruses and Pseudoviruses...........................................................136 3.4.5. Multiple-cycle Infection Assays..................................................137 3.4.6. Single-cycle Infection Assays......................................................138 3.4.7. Flow Cytometry...........................................................................139 3.4.8. Time-of-addition Assay...............................................................140 3.5. Results..........................................................................................................141 3.5.1. Dichotomous Sensitivity to PSC-RANTES in Multiple- and......141 Single-Cycle Assays 3.5.2. Dichotomous Multiple- and Single-Cycle Sensitivity.................145 Differences are Specific to PSC-RANTES 3.5.3. Prolonged Incubation with PSC-RANTES Recapitulates...........146 Multiple-Cycle Assays 4 3.5.4. Inhibition of HIV-1 Replication by PSC-RANTES with a..........150 Downregulation-defective Variant of CCR5 3.5.5. Multiplicity of Infection Effects on PSC-RANTES IC50 Value..153 3.5.6. Multiplicity of Infection Effects in Wild Type and M7-CCR5...160 3.5.7. Kinetics of PSC-RANTES Inhibition of Entry............................160 3.6. Discussion...................................................................................................166 Chapter 4. Intrinsic Sensitivity of Human Immunodeficiency Virus Type 1.................171 Isolates to PSC-RANTES is Mediated by Differential CCR5 Affinity 4.1. Preface..........................................................................................................172 4.2. Abstract........................................................................................................173 4.3. Introduction..................................................................................................174 4.4. Materials and Methods.................................................................................177 4.4.1. Reagents.......................................................................................177 4.4.2. Plasmids.......................................................................................177 4.4.3. Cell Lines.....................................................................................177 4.4.4. Pseudovirus Production and Single-Round Entry Assays...........178 4.4.5. Incorporation of Cleaved gp120 into Virions..............................178 4.4.6. Infection of 293 Cells with Inducible Levels of CCR5...............179 and CD4 4.4.7. Flow Cytometry...........................................................................180 4.4.8. Kinetic Entry Assay.....................................................................181 4.5. Results..........................................................................................................182 4.5.1. Neutralization of V3 Crown Mutants by sCD4...........................182 5 4.5.2. Effects of CD4 and CCR5 Expression Levels on Infectivity......185 of V3 Crown Polymorphisms 4.5.3. Infectivity Impact of Dominant Negative Hetero-.......................191 oligomerization on V3 Crown Polymorphisms 4.5.4. V3 Crown Polymorphisms Impact on Fusion Kinetics................195 4.6. Discussion...................................................................................................199 Chapter 5. General Discussion........................................................................................206