HIV-1 INTERSUBTYPE RECOMBINATION WITHIN GP120 IMPOSES SEVERE FUNCTIONAL RESTRICTION ON RESULTANT ENVELOPES by BERNARD SSENTALO BAGAYA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisors: Yong Gao, MD, PhD. Eric J. Arts, PhD. Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY MAY, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of BERNARD SSENTALO BAGAYA __________________________________________________________ Doctor of Philosophy candidate for the __________________________________________ degree*. David McDonald, Ph.D. Signed _______________________________________________________ (chair of the committee) Jonathan Karn, Ph.D. _______________________________________________________ Eric J. Arts, Ph.D. _______________________________________________________ Yong Gao, M.D, PhD. _______________________________________________________ (dissertation advisor) Neil Greenspan, M.D, PhD. _______________________________________________________ Carlos Subauste, M.D, PhD. _______________________________________________________ October 24, 2014 (date) _______________________________________________________ *We also certify that written approval has been obtained for any proprietary material contained therein 1 TABLE OF CONTENTS Content Page Approval Signatures…………………………………………................................. 1 Table of contents……………...…………………………....…………………........ 2 Dedication……………………..…………………………………………..……… 8 Acknowledgements…..……………………………………..…………..………… 9 List of figures………………………………………………………………..……. 11 List of tables………………………………………….……………..…………...... 14 List of Appendices……………………………………………..………………..... 15 List of abbreviations…………………………….…….………………………….. 16 Abstract…………………………………….…………………………..…………. 21 CHAPTER ONE: INTRODUCTION……………………….…..………………... 23 1.1. History of HIV-1 and AIDS…………………………………...... 23 1.2. The HIV-1 particle………………..……….……………...…….. 25 1.3. The HIV-1 genome and proteins…………………………..……. 25 1.3.1. Regulatory genes/elements………...…….…………. 27 1.3.1.1. The LTR sequences..……………….……... 27 1.3.1.2. Tat/rev……………………………...…….... 28 1.3.2. Accessory genes (vif, vpr, vpu, and nef)………...…. 28 1.3.3. The HIV-1 structural genes (gag, pol and Env)....…... 29 1.3.3.1. gag……………………….…………….….. 29 2 1.3.3.2. pol…………………………......................... 30 1.3.3.3. The Env gene……………………................ 31 1.4. The HIV-1 Envelope glycoprotein…………………..…..…….… 34 1.5. The HIV-1 life cycle………………………....……………..……. 38 1.5.1. HIV-1 entry………………………...………………. 38 1.5.2. Reverse transcription…………………..................... 43 1.5.3. Assembly, budding and maturation…………...…… 45 1.6. HIV-1 diversity…………………………..……………………….. 50 1.6.1. HIV-1 recombination………...…….…….................. 50 1.6.1.1. Models of HIV-1 recombinations…... 52 1.6.1.2. Consequences of HIV-1 intersubtype recombination…….............................. 59 CHAPTER TWO: MATERIALS AND METHODS……………………...……… 66 2.1. HIV-1 primary isolates………………….…........................................... 66 2.2. Mammalian cells………………….....…………………………......…... 66 2.3. Yeast and bacteria cell lines…………………………….…..………..… 67 2.4. Reagents and Media………………………...………..………………… 67 2.5 . Preparation of culture media and plates……..…..................................... 68 2.6 . Yeast gap repair homologous recombination-based cloning method….. 68 2.7 . Phenol:chloroform:Isoamyl alcohol plasmid DNA extraction from yeast………...…..……......…………………………………………... 69 3 2.8 . Bacterial transformation and plasmid extraction……............................. 70 2.9 . The complementation system for production of HIV-1 in-vitro; the pREC_nfl_HIV and the pCMV_5’LTR_cplt plasmids........................ 71 2.10 . Transfection and infection for virus propagation...……………...……. 72 2.11 . Reverse Transcriptase-activity assay (RT-Assay)……………..……... 73 2.12 . In-house gp140/160 ELISA…………………………..………………. 74 2.13 . Veritrop assay………………………………………...……………...... 75 2.14 . Production of chimeric HIV-1………………………………………... 77 2.15 . The Dual infection method for production of HIV-1 recombinants.... 79 2.16. Design and construction of the HIV-1 Env recombination system.…... 80 2.17 . HIV-1 Env cloning……………………………………………………. 81 2.18 . Clonal sequencing………………….…………………………………. 82 2.1 9. Downloading HIV-1 recombinant Env sequences from database.......... 83 2.20 . Design and construction of the HIV-1 pol recombination system......... 84 2.21 . 454 sequencing…………..….………………………............................ 86 CHAPTER THREE: A NOVEL SYSTEM FOR GENERATION OF PURE AND FUNCTIONAL HIV-1 POL AND ENV INTERSUBTYPE RECOMBINANTS IN VITRO………...…………………………......................... 89 3.1. PREFACE………………………………………………..………. 90 3.2. INTRODUCTION………………………………………..………. 92 3.3. RESULTS…………………………….………………..………… 94 3.3.1. Establishment of the yeast based recombination/HIV-1 cloning system..................................................................... 94 3.3.2. The complementation system for production of chimeric infectious HIV-1 in cell cultures………………................. 96 4 3.3.3. Establishment of the HIV-1 Env recombination system….. 103 3.3.4. Production of replicating intersubtype A/D Envelope recombinant HIV-1 by the Env recombination system…... 104 3.3.5. Optimization of the HIV-1 intersubtype Env recombination system…………………………………….. 110 3.3.6. The HIV-1 intersubtype Env recombination system produces 100% recombinants……………………………. 113 3.3.7. The HIV-1 pol intersubtype recombination system………. 117 3.3.8. The pol recombination system produces infectious replicating virus…………………………………………... 118 3.4. Discussion…………………………………….………………..... 122 CHAPTER FOUR: RECOMBINATION BREAKPOINTS IN GP120 IMPOSE SEVERE FUNCTIONAL RESTRICTION ON RESULTANT ENVELOPE GLYCOPROTEINS………………………………..…………......... 126 4.1. PREFACE……...……...…………….…………………………... 127 4.2. INTRODUCTION……………………………..………................ 128 4.3. RESULTS……………………………….…………..……...……. 131 4.3.1. Production of HIV-1 Env recombinants using the novel Env recombination system that enriches for functional recombinants……………………………………………… 131 4.3.2. Clonal sequence analysis shows an absence of gp120 recombination breakpoints in recombination system virus population…………..…………………………………….. 136 4.3.3. The dual infection method generates gp120 breakpoints, ruling out lack of adequate sequence similarity to support homologous recombination…............................................. 138 4.3.4. gp120 breakpoints are present in sequence database- 5 derived recombinant Envs, overwhelmingly concentrated in the C1 domain and very few elsewhere………………... 139 4.3.5. Pyro-sequencing shows that HIV-1 Env recombination system is not defective in generating gp120 breakpoints… 143 4.4. DISCUSSION………………...…………….…………………… 147 4.5. CONCLUSION………………………………………………….. 150 CHAPTER FIVE: BOTTLENECKS FOR GENERATION OF FUNCTIONAL HIV-1 ENV RECOMBINANTS IN VITRO……………………. 151 5.1. PREFACE……………...…….………..…………….................... 152 5.2. INTRODUCTION………………….………………..................... 153 5.3. RESULTS………………………...………….…………………... 155 5.3.1. Generation of Intersubtype A/D gp120 recombinants….... 155 5.3.2. Full length HIV-1 A/D recombinant Envelopes with breakpoints in gp120 are non-functional when cloned into a neutral NL4-3 backbone…………...…………..…...…... 156 5.3.3. Replacement of HIV-1 NL4-3 tat1/rev1 sequence with D109 tat1/rev1 restores function in 2 of the 21 EnvA/D recombinants……………………………………………… 161 5.3.4. Cloning only gp120 region does not overcome the functional restriction of EnvA/Ds with breakpoints in gp120……………………………………………………... 162 5.3.5. Re-insertion of NL4-3 gp120 C1 and C5 domains back into non-functional recombinant gp120 chimeric plasmids restored function in 4 of 9 recombinants……..… 163 5.4. DISCUSSION……………………………….………………… 171 CHAPTER SIX: GENERAL CONCLUSIONS AND FUTURE DIRECTIONS... 176 6 6.1. General conclusions……….…...……………………...…...…... 176 6.2 . Future direction…..………………………...…………...…….... 178 6.2. 1. Study on HIV-1 intersubtype recombinant Envelope function restriction factors................................................ 178 6.2.2. Study on anti-HIV-1 drug resistance……….….…..….... 179 6.2.3. Development of anti-HIV-1 vaccines with intersubtype Env recombinants...…..………………………………… 181 APPENDICES………………………...……………….………………................ 183 REFERENCES…………………………….…………………...………………... 193 7 DEDICATION This thesis is dedicated to my darling wife Lillian L. Bagaya. You have been my strength in times of weakness and my guiding star when all seemed dark. You made the old English proverb of “behind every successful man, there is a woman”, come to reality. To you I am forever indebted. 8 ACKNOWLEDGEMENTS To my father who, despite of having had no opportunity to receive an education, knew its value, toiled and unreservedly spent his hard earned income to pay for my education so I wouldn’t have to earn a living through hard labor like he did; you are my hero. To my mother who has been a never drying fountain of encouragement and wisdom, I will always revere you. Great thanks to my wife Lillian and to my little Angels Daniella, Diella and Atuwaire for the sacrifices you have made for us to achieve this. You have been patient and encouraging, I have almost been missing in your lives but you endured, thank you for understanding and persevering. I thank all the current and former members of the Arts/Gao laboratories at CWRU School of Medicine for the help, friendship and humor we shared. It would have been a lot harder without you around. I am grateful