RNA interference, understanding the collateral effects of HIV-1 and ZIKV Sergio Paulo Alpuche Lazcano Department of Medicine Division of Experimental Medicine McGill University, Montréal, Québec, Canada December, 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy. © Sergio Paulo Alpuche Lazcano, 2019 Table of Contents LIST OF FIGURES ........................................................................................................................ 7 LIST OF TABLES .......................................................................................................................... 8 ABSTRACT .................................................................................................................................. 10 RÉSUMÉ ...................................................................................................................................... 12 ACKNOWLEDGMENTS ............................................................................................................ 14 LIST OF ABBREVIATIONS ....................................................................................................... 16 CONTRIBUTION TO ORIGINAL KNOWLEDGE ................................................................... 24 CONTRIBUTION OF AUTHORS............................................................................................... 25 CHAPTER I .................................................................................................................................. 27 INTRODUCTION ................................................................................................................... 27 1.1 RNA interference (RNAi) ................................................................................................... 28 1.1.1 RNAi discovery and general characteristics ................................................................ 28 1.1.2. The canonical miRNA biogenesis ................................................................................ 29 1.1.3 The loading RISC.......................................................................................................... 32 1.1.3.1 The loading RISC: Dicer ........................................................................................... 32 1.1.3.2 The loading RISC: TRBP ........................................................................................... 35 1.1.3.3 The loading RISC: Ago2 ............................................................................................ 37 1.1.4 MiRNA strand selection and pairing ............................................................................ 38 1.1.5 P-bodies ........................................................................................................................ 39 1.2 Human immunodeficiency virus........................................................................................ 43 1.2.1 HIV Discovery, origins, and contemporary impacts .................................................... 43 1.2.2 HIV-1 genome and virion structure .............................................................................. 46 1.2.3 The HIV-1 replication cycle and Gag protein .............................................................. 49 1.2.3.1 Virus binding, entry, and the reverse transcription................................................... 51 1.2.3.2 Integration and transcription .................................................................................... 52 1.2.3.3 Gag, viral assembly, release and maturation. ........................................................... 54 1.2.4 HIV-1 infection ............................................................................................................. 57 1.2.4.1 Acute infection and the founder virus. ....................................................................... 59 1.2.4.2 Chronic infection and AIDS. ..................................................................................... 60 1.2.5 Current ART ................................................................................................................. 61 1 1.2.6 Latency and viral reservoir .......................................................................................... 62 1.2.7 HIV-1 cure strategies.................................................................................................... 63 1.3 Zika virus ............................................................................................................................ 66 1.3.1 Emergence of Zika virus ............................................................................................... 66 1.3.2 ZIKV and genome ......................................................................................................... 68 1.3.3 ZIKV replication cycle .................................................................................................. 71 1.3.3.1 Binding, entry, fusion and release ............................................................................. 71 1.3.3.2 Translation, polyprotein cleavage and RNA replication ........................................... 71 1.3.3.3 Assembly and release................................................................................................. 72 1.3.4. ZIKV phylogenomics and pathogenesis ....................................................................... 75 1.3.5. ZIKV transmission ....................................................................................................... 76 1.3.6 ZIKV pathology............................................................................................................. 78 1.3.6.1 CZVS, neurologic abnormalities ............................................................................... 79 1.3.6.2 CZVS, ocular and other abnormalities ...................................................................... 79 1.3.7. ZIKV, potential treatments and vaccines .................................................................... 80 1.4. RNAi and viruses interplay, HIV-1 and ZIKV ................................................................ 82 1.4.1 miRNAs and HIV-1 ....................................................................................................... 83 1.4.1.1 Encoded vmiRNAs by HIV-1...................................................................................... 83 1.4.1.2 miRNAs that directly target HIV-1 RNA.................................................................... 84 1.4.1.3 miRNAs that impair HIV-1 through host dependency factors ................................... 85 1.4.1.4 miRNAs that promote HIV-1 replication ................................................................... 86 1.4.2 RNAi proteins and HIV-1 ............................................................................................. 87 1.4.3 miRNAs and ZIKV ........................................................................................................ 91 1.5 REFERENCES .................................................................................................................. 93 1.6 Rationale and hypothesis ................................................................................................. 122 1.6.1 Objectives ....................................................................................................................... 122 1.6.2 References ................................................................................................................... 124 CHAPTER II ............................................................................................................................... 125 HIV-1 Gag interacts with Dicer and increases its binding to specific microRNAs. ........ 125 2.1 PREFACE ........................................................................................................................ 126 2.2 ABSTRACT ..................................................................................................................... 128 2.3 INTRODUCTION ........................................................................................................... 129 2 2.4 RESULTS ......................................................................................................................... 132 2.4.1 RNA interference remains functional in cells expressing HIV-1 ................................ 132 2.4.2 HIV-1 expression in human cells does not change the localization of RISC proteins in the cytoplasm ....................................................................................................................... 134 2.4.3 Viral Gag colocalizes with Dicer in HIV-1 producing cells ...................................... 136 2.4.4 Dicer and HIV-1 Gag interaction is independent of RNA .......................................... 138 2.4.5 Dicer and Gag are in close proximity in both the nucleus and cytoplasm ................. 139 2.4.6 Dicer catalytic activity on pre-miRNA Let7c and pre-miRNA29a is maintained in the presence of Gag ................................................................................................................... 141 2.4.7 Dicer/Gag interaction results in higher binding of specific miRNAs. ........................ 142 2.4.8 Increased binding of miR642a 3p, miR766 5p and miR766 3p to Dicer when HIV-1 Gag is co-expressed. ............................................................................................................ 145 2.4.9 HIV-1 interacting