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Targeting Viral RNA Processing to Control HIV-1 Infection

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Targeting Viral RNA Processing to Control HIV-1 Infection Targeting Viral RNA Processing to Control HIV-1 Infection by Raymond Waiman Wong A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Raymond Waiman Wong (2018) Targeting Viral RNA Processing to Control HIV-1 Infection Raymond Waiman Wong Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto 2018 Abstract Resistance to current drugs requires innovative strategies to control HIV-1 infection. We aimed at developing novel inhibitors of HIV-1 replication by targeting viral RNA processing—a stage dependent on conserved host proteins. We identified 12 members of the digoxin family (cardiotonic steroids, CSs) that impede HIV growth in HIV-infected PBMCs from patients at concentrations (IC50s: 1.1-1.3 nM) that were 2-26 times below those used to treat people with heart conditions. I hypothesized that the response to CSs was due to activation of intracellular signaling pathways initiated upon binding to the Na+/K+-ATPase. CSs suppressed HIV-1 gene expression in part through MEK1/2-ERK1/2 activation, independent of the Ca2+-overloading mechanism responsible for their toxicity. Supporting this hypothesis, Na+/K+-ATPase depletion and MEK1/2-ERK1/2 activator addition also inhibited HIV-1 expression. All CSs tested induced oversplicing of HIV-1 RNAs, reducing unspliced (Gag) and singly spliced RNAs (Env/p14-Tat) encoding critical HIV-1 structural/regulatory proteins. Consequently, all CSs induced nuclear retention of genomic/unspliced viral RNAs, supporting viral RNA processing as the underlying mechanism disrupting HIV-1 replication. Concomitantly, CSs induced modification of SRp20 and Tra2β (whereas digitoxigenin caused de-modification of these splicing factors), which could ii account for responses observed. Consistent with this hypothesis, overexpression of SRp20 altered HIV-1 RNA processing in a similar manner as CS addition. In expanding this approach to other viruses dependent on RNA processing, we identified the splicing-modulator 5342191 as both an inhibitor of HIV-1 and Adenovirus replication (IC50: 750 and 900 nM, resp.). In contrast to CSs, 5342191 inhibition of HIV-1 expression also included proteasomal degradation of Tat, low perturbation of host gene expression (<0.5%), and G protein signaling through Ras-Raf- MEK1/2-ERK1/2 from the cell membrane but not p38 MAPK or Ca2+ flux. Overexpression of Ras small-G proteins phenocopied 5342191’s effects. These studies support modulating viral RNA processing and targeting alternative cellular targets for controlling HIV-1/Adenovirus infection(s). iii Acknowledgments I thank my supervisor, Dr. Alan Cochrane, for providing me the great opportunity to study these interesting projects, where I found my utmost passion for science. I am very grateful for his guidance and knowledge over the course of this dissertation. I would also like to express my greatest appreciation to my supervisory committee members: Scott Gray-Owen, Jeffrey Lee, and co-supervisor Clifford A. Lingwood, for their generous time in providing advice, encouragement, and reagents/equipment. I couldn’t have done this without your support. I thank the Canadian Institutes of Health Research (CIHR) for selecting me for their Doctoral Award – Frederick Banting and Charles Best Canada Graduate Scholarship, UofT/LMP for a couple of fellowship/awards, SGS for a couple of conference/travel grants, and conference committees that awarded me scholarships to attend the Canadian Association for HIV Research (CAHR) conferences for 2012 and 2015 and HIV DART™: Frontiers in Drug Development in Antiretroviral Therapies conference of 2014. Such gatherings have immensely grown my knowledge, helped me create amazing connections, and let me enjoy the fun of science! I thank all past and present members of the Cochrane lab, such as martial-arts Master Dr. Simon Duffy, Dr. Liang “The Panda or Cookie Monster” Ming, and Dr. Alex T.Y. Chen for their critical thoughts on my projects, hardworking undergraduate students such as Annie Y.Q. Mao, Chathura Wijewardena, Lewis Liu, Melissa Geng, etc., Dr. Rade Sajic for a good Northern blot protocol, and other labmates, especially ones who brought positivity and entertainment to the lab. I also wish to thank Shariq Mujib for training me on preparing HIV infected PBMCs for culture, Wendy Dobson-Belaire for teaching me how to perform HIV-1 infections of PBMCs, principal investigators who provided invaluable reagents for our studies (see manuscripts) such as Dr. Peter Stoilov for SMN2 compounds and Mario Ostrowski for PBMCs, many researchers who donated their time to proofread my written materials (i.e. Segen Kidane, see manuscripts), Natasha Christie and Shannon McGraw for their managerial advice, Ferzeen Sammy for assistance as our mommy on solving graduate student account issues, Dr. Harry Elsholtz, Donald R. Branch, and Dr. Tram N.Q. “Boss” Pham for their superb encouragement and advice, and Dr. Mark Wainberg for his motivating speeches and project support (who will be dearly missed!). I thank my parents, William S. and Susan Wong, for their support and raising us with everything we ever needed. I am also indebted to many friends, including Tiffany Pedron (who also contributed to many of my food porfolios entitled “Antidepressants”, “Nom Noms”, “Oinkings”, and “To Eat or Not to Eat, that is Not a Question”), Kaustabh “Bunty” Singh, Norman Ng, Khalid and Nicola Qureshi, Mark Ng, and many others, who have supported and encouraged me since the beginning. In further helping me maintain my sanity throughout all of these years, I thank members of my sports teams as follows: LMP volleyball team (Alex Falkenhagen, Stephen McCarthy, Olga Brashavitskaya, etc. which won a 2012-13 UofT Intramural championship and a free T-shirt), dodgeball team (Drs. Atta Goudarzi, Steven Hersch, Karen Founk, Fiona Coutinho, Shicong Betty Zou, Joe Bondy-Denomy, etc. which won over 7 championships and T-shirts) in the TSSC between 2009-2015 and in Nation Leagues (runner up in one tournament once so far) between 2016-2018, and LMP softball team, pick-up soccer/basketball, and events organized every year. Without the tremendous support from all of these people, organizations, sport teams, family, and friends, I could never have reached this milestone. Thank you everyone for being there! iv Table of Contents Acknowledgments ........................................................................................................................ iv Table of Contents ...........................................................................................................................v List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................ xvii List of Abbreviations (continued) .............................................................................................. xviii List of Abbreviations (continued) ................................................................................................ xix 1 Introduction .............................................................................................................................20 1.1 The HIV pandemic .............................................................................................................20 1.2 Vaccines, cures, and treatments to control the spread and infection of HIV-1 .................22 1.3 HIV-1 RNA processing ......................................................................................................25 1.3.1 Control of HIV-1 transcription by Tat ...................................................................27 1.3.2 RNA processing of host and HIV-1 transcripts .....................................................28 1.3.3 Regulation of HIV-1 RNA splicing .......................................................................29 1.3.4 Regulation of HIV-1 gene expression by Rev .......................................................33 1.4 Role of host splicing factors in the cell and in HIV-1 replication .....................................35 1.4.1 RNA processing in human diseases .......................................................................35 1.4.2 hnRNP functions ....................................................................................................35 1.4.3 SR protein functions ..............................................................................................36 1.4.4 Effect of modulating host splicing factors on HIV-1 replication ..........................36 1.5 Regulation of SR protein functions by phosphorylation and other post-translational modifications......................................................................................................................37 1.5.1 Effect of different post-translational modifications on SR protein function .........37 1.5.2 Role of phosphorylation on SR proteins ................................................................38 v 1.6 Regulation of host alternative RNA splicing by intracellular SR-protein/signaling kinases and their secondary messengers ............................................................................39
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