MECHANISMS OF CCR5 AGONIST/ANTAGONIST INHIBITION OF HIV-1 ENTRY AND IN VITRO SELECTION OF VIRUS RESISTANT TO MARAVIROC by ANNETTE NICOLE RATCLIFF 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 January, 2013 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Annette Nicole Ratcliff _______________________________________________________________________ Doctor of Philosophy candidate for the __________________________________________ degree*. Jonathan Karn, Ph.D. (signed)_______________________________________________________ (chair of the committee) David McDonald, Ph.D. _______________________________________________________ Scott Sieg, Ph.D. _______________________________________________________ John C. Tilton, M.D. _______________________________________________________ Eric J. Arts, Ph.D. _______________________________________________________ (dissertation advisor) October 9, 2012 (date) _______________________________________________________ *We also certify that written approval has been obtained for any proprietary matieral contained therein. 0 TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................1 LIST OF TABLES ...........................................................................................................6 LIST OF FIGURES .........................................................................................................7 ACKNOWLEDGEMENT ...............................................................................................10 LIST OF ABBREVIATIONS ..........................................................................................11 ABSTRACT .....................................................................................................................13 1. CHAPTER 1: INTRODUCTION .............................................................................15 1.1 HIV and AIDS ..............................................................................................16 1.2 HIV-1 Classification and Diversity ..............................................................16 1.3 HIV-1 Genome and Proteins ........................................................................18 1.4 HIV Virion ...................................................................................................22 1.5 HIV Replication Cycle .................................................................................23 1.5.1 Viral Entry and Cellular Tropism ................................................23 1.5.2 Reverse Transcription ..................................................................26 1.5.3 Nuclear Import and Integration ....................................................28 1.5.4 RNA Transcription and Export ....................................................29 1.5.5 Assembly and Packaging .............................................................31 1.5.6 Viral Budding, Release, and Maturation......................................32 1.6 Antiretrovirals and Drug Resistance ............................................................33 1.7 Molecular Aspects of HIV Entry ..................................................................36 1.7.1 Envelope Structure .......................................................................36 1 1.7.2 CD4 and Interactions with gp120 ................................................40 1.7.3 Chemokine Receptors and Interactions with gp120 ....................41 1.7.4 Gp41 Mediated Membrane Fusion ..............................................44 1.8 Entry Inhibitors and Mechanisms of Resistance ..........................................46 1.8.1 Attachment Inhibitors ..................................................................46 1.8.2 Fusion Inhibitors .........................................................................50 1.8.3 Inhibitors of gp120-CXCR4 Interaction ......................................51 1.8.4 Inhibitors of gp120-CCR5 Interaction .........................................52 1.8.4.1 Chemokine Analog Inhibitors ....................................53 1.8.4.2 Small Molecule Antagonists ......................................54 1.8.4.3 Resistance to CCR5 Inhibitors ...................................56 1.9 Summary of Thesis Work .............................................................................82 2 CHAPTER 2: MECHANISTIC STUDIES OF HIV-1 ENTRY INHIBITORS REVEALS DIFFERENTIAL SENSITIVITY TO PSC-RANTES INHIBITION INVOLVES COMPETITIVE CCR5 BINDING .....................................................84 2.1 Preface ..........................................................................................................85 2.2 Abstract ........................................................................................................86 2.3 Introduction ..................................................................................................88 2.4 Materials and Methods .................................................................................91 2.4.1 Cells, Viruses and Inhibitors .......................................................91 2.4.2 Plasmids ......................................................................................93 2.4.3 Reverse Transcriptase Activity Assay ........................................94 2.4.4 Multiple-Cycle Drug Susceptibility Assays................................94 2 2.4.5 Single-Cycle Drug Susceptibility Assays ...................................95 2.4.6 Flow Cytometry ..........................................................................95 2.5 Results ..........................................................................................................97 2.5.1 Dichotomous Sensitivity to PSC-RANTES in Multiple and Single- Cycle Assays ................................................................................97 2.5.2 Inhibition of HIV-1 Replication with a Downregulation-Defective Mutant of CCR5 ...........................................................................98 2.5.3 Prolonged Incubation with PSC-RANTES Recapitulates Multiple- Cycle Assays ................................................................................100 2.5.4 PSC-RANTES Inhibits HIV Entry by Competitive CCR5 Binding ......................................................................................................102 2.5.5 Kinetics of PSC-RANTES Inhibition of HIV Entry ....................103 2.6 Discussion ....................................................................................................112 3 CHAPTER 3: HIV-1 RESISTANCE TO MARAVIROC CONFERRED BY A CD4 BINDING SITE MUTATION IN THE ENVELOPE GLYCOPROTEIN GP120 ..117 3.1 Preface ..........................................................................................................118 3.2 Abstract ........................................................................................................119 3.3 Introduction ..................................................................................................120 3.4 Materials and Methods ..................................................................................124 3.4.1 Cells, Viruses and Inhibitors ........................................................124 3.4.2 Resistant Virus Isolation ..............................................................124 3.4.3 Multiple-Cycle Drug Susceptibility Assays.................................125 3.4.4 Construction of Gp120 Chimeric Viruses....................................125 3 3.4.5 Oligonucleotide Ligation Assay ..................................................126 3.4.6 Site-Directed Mutagenesis PCR Method .....................................126 3.4.7 In Vitro Fitness Assays ................................................................127 3.4.8 Structural Modeling .....................................................................128 3.5 Results ...........................................................................................................129 3.5.1 HIV-1 Primary Isolates Demonstrate Variable Sensitivity to Maraviroc .....................................................................................129 3.5.2 Maraviroc Resistant Mutant Generated in Cell Culture ..............129 3.5.3 MVC.21 Cross Resistant to another CCR5 Antagonist ...............131 3.5.4 Multiple Mutations Throughout Gp120 in Resistant Virus .........132 3.5.5 Sensitivity of Selected A74 HIV-1 Clones to MVC Inhibition ...135 3.5.6 Relative Fitness of MVC-Resistant and Sensitive HIV-1 Clones ......................................................................................................136 3.5.7 K425 is Primary Resistance Mutation .........................................137 3.5.8 Shift in IC50 Versus the MPI Effect Denotes MVC Resistance .137 3.5.9 Model of K425 in Gp120 Structure Suggests Role in CD4 Binding Affinity .........................................................................................138 3.6 Discussion ......................................................................................................153 4 CHAPTER 4: ENHANCED CD4 BINDING AFFINITY NOVEL MECHANISM OF HIV-1 RESISTANCE TO MARAVIROC ..........................................................158 4.1 Preface............................................................................................................159