Selective Induction of Apoptosis in HIV-infected Macrophages by Simon Xin Min Dong A thesis submitted in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Microbiology and Immunology Department of Biochemistry, Microbiology, and Immunology Faculty of Medicine University of Ottawa © Simon Xin Min Dong, Ottawa, Canada, 2020 Abstract The eradication of Human Immunodeficiency Virus (HIV) from infected patients is one of the major medical problems of our time, primarily due to HIV reservoir formation. Macrophages play important roles in HIV reservoir formation: once infected, they shield HIV against host anti-viral immune responses and anti-retroviral therapies, help viral spread and establish infection in anatomically protected sites. Thus, it is imperative to selectively induce the apoptosis of HIV-infected macrophages for a complete cure of this disease. I hypothesize that HIV infection dysregulates the expression of some specific genes, which is essential to the survival of infected host cells, and these genes can be targeted to selectively induce the apoptosis of HIV-infected macrophages. My objective is to identify the genes that can be targeted to eradicate HIV reservoir in macrophages, and to briefly elucidate the mechanism of cell death induced by targeting one of the identified genes. A four-step strategy was proposed to reach the goal. First, 90k shRNA lentivirus pool technology and microarray analysis were employed in a genome-wide screen of genes and 28 promising genes were found. Second, siRNA silencing was applied to validate these genes with 2 different HIV-1 viruses; as a result, 4 genes, Cox7a2, Znf484, Cdk2, and Cstf2t, were identified to be novel gene targets. Third, the 4 validated genes were characterized by literature review. Lastly, I briefly elucidated the mechanism of apoptosis induced by targeting one of the identified gene, Cox7a2. The results of this study can help to understand the mechanisms of HIV reservoir formation in macrophages and propose promising therapeutic targets for the eradication of HIV-infected macrophages from patients. II Acknowledgements First of all, I would like to thank my supervisor, Dr. Ashok Kumar, for his mentorship of my PhD studies. It was his diligence, intelligence, and full support that made me step forward day by day. This study was also under the instructions of Dr. Franco Vizeacoumar from the University of Saskatchewan; his timely and patient guidance made applying a novel technology to this research possible. Dr. Angela Crawley, Dr. Tommy Alain, and Dr. Jonathan Angel, who were my Thesis Advisory Committee members, also gave me pertinent advice during this study; their encouragement allowed me to grow as an independent medical researcher. In addition, Xiao Xiang, Niranjala Gajanayaka, Ramon Caballero, and Hamza Ali provided me everyday assistance. Duale Ahmed, David L. F. Roy, and Faria Ahmed also helped for some experiments and data analysis. Dr. Sandra Côte, Stephanie B. Schinkel, and Teslin Sandstrom at OHRI provided me assistance whenever I needed. Dr. Jason Fernandes, and Dr. Hapsa Mamady were my role models to be a medical researcher. Both Jasmine Huang and Ana Radar worked as part-time summer students for this project. Finally, I would also like to say many thanks to Lynn Kelly, Martine St-Jean, Stephen Baird, Keith Wilson, and many other staff at the Apoptosis Research Center of Children’s Hospital of Eastern Ontario. It was their timely assistance that made my work highly efficient so that I could progress rapidly. The selfless contributions from Katelynn J. Rowe (OHRI) were also indispensable to complete this research project. Here I would like to express my sincere gratitude and respects to all of them. III Table of Contents Abstract .................................................................................................................... II Acknowledgements ................................................................................................ III Table of Contents ................................................................................................... IV List of Abbreviations ............................................................................................ VIII List of Figures ........................................................................................................ XI List of Tables ........................................................................................................ XIII 1. General Introduction ........................................................................................... 1 1.1 A brief history on HIV/AIDS ........................................................................... 1 1.2 General information about HIV/AIDS ............................................................ 2 1.3 Transmission, infection, integration, and life cycle of HIV ........................ 6 1.4 Immune dysregulation due to massive depletion of CD4+ T cells ............ 9 1.5 The natural course of HIV infection without treatment ............................. 14 1.6 Anti-retroviral treatments after HIV infection ............................................ 16 1.7 Genetic diversity of HIV viruses ................................................................. 18 1.8 HIV reservoir formation after infection ...................................................... 19 1.9 The interplay between HIV and macrophages ........................................... 27 1.10 HIV infection dysregulates the gene expression of macrophages ........ 28 1.11 Roles of HIV proteins in apoptosis ........................................................... 30 1.12 Mitochondria in apoptosis of HIV-infected macrophages ...................... 33 1.13 Vpr-induced apoptosis of macrophages ................................................. 34 1.14 Selective induction of apoptosis in HIV-infected macrophages ............ 36 1.15 Targeting IAPs may induce apoptosis of HIV-infected macrophages ... 37 1.16 90k shRNA lentivirus pool for a genome-wide screen of target genes . 38 1.17 Approaches of targeting genes in primary human macrophages ......... 42 1.18 HIV viruses for in vitro infection of primary human macrophages ......... 44 2. Rationale ............................................................................................................ 49 3. Hypothesis ......................................................................................................... 49 4. Objectives .......................................................................................................... 49 5. Materials and Methods ...................................................................................... 50 5.1 Cell culture and differentiation of U937, THP-1, and U1 cell lines ........... 50 IV 5.2 Preparation of primary human MDMs and M1 polarization ...................... 50 5.3 HIV-1 production, in vitro infection of MDMs and HIV-p24 ELISA ............ 51 5.4 Preparation of 90k shRNA lentivirus pool ................................................. 52 5.5 Infecting myeloid lineage cells with 90k shRNA lentivirus pool .............. 52 5.6 Preparation of HIV-eGFP and HIV-HSA viruses from plasmid DNA ........ 53 5.7 Infection of primary MDMs with HIV-eGFP and HIV-HSA ......................... 54 5.8 siRNA transfection for transfection reagent selection ............................. 55 5.9 Detection of siRNA transfection rates ....................................................... 56 5.10 Monitoring cell viability and GFP expression.......................................... 56 5.11 Localization of transfected siRNA in primary human MDMs ................. 57 5.12 Flow cytometry analysis of PI staining and GFP expression................. 57 5.13 siRNA transfection of primary human MDMs for validation ................... 58 5.14 siRNA transfection of respirasome complex genes ............................... 58 5.15 Apoptosis of HIV-infected MDMs induced by siRNA silencing .............. 59 5.16 Propidium Iodide (PI) Staining to evaluate cell death ............................. 59 5.17 Isolation of HIV-HSA-infected MDMs ........................................................ 60 5.18 Analysis of caspase activation ................................................................. 60 5.19 TNF-α ELISA and cytokine ELISA array ................................................... 60 5.20 SDS-PAGE and Western blotting analyses.............................................. 61 5.21 Apoptosis of HIV-infected MDMs induced by chemical inhibitors ........ 62 5.22 ROS production detection ........................................................................ 63 5.23 Microscopy ................................................................................................. 64 5.24 Statistical analysis ..................................................................................... 64 5.25 Ethics statement ........................................................................................ 64 6. Results ............................................................................................................... 65 Chapter 6.1: Optimizing siRNA transfection into primary human macrophages by reversing Resveratrol-induced apoptosis ..................................................... 65 1. Introduction ................................................................................................. 65 2. Results .......................................................................................................