Investigating early events in HIV-1 replication: the role of envelope signalling and PAF1 restriction factor Ana Guerrero Alonso A thesis submitted in partial fulfillment of the requirements of the Degree of Doctor of Philosophy 2015 Centre for Immunology and Infectious Disease The Blizard Institute Barts and The London School of Medicine and Dentistry Queen Mary, University of London 1 Abstract To enter cells, HIV utilises the envelope (Env) protein to engage the CD4 receptor and a co-receptor, CXCR4 or CCR5. In addition to its role in HIV entry, the Env protein also triggers intracellular signalling events. Expression of the HIV- 1 and HIV-2 Env gp41 cytoplasmic tails triggered a significant decrease in microtubule acetylation at the single cell level. Since microtubule stability is controlled by Rac1, the role of Rac1 in HIV-1 infection was investigated. Treatment of TZM-bl cells with a Rac1 inhibitor that blocks its upstream activators, Tiam1 and TrioN, inhibited HIV-1 NL4.3 (X4-tropic) but not HIV-1 BaL (R5-tropic) suggesting a role for these activators which is possibly, co-receptor dependent. Subsequently, an siRNA screen including 145 upstream regulators of Rac1, cdc42 and RhoA were investigated in their role in HIV-1 NL4.3 and HIV-1 BaL infection. As a result, 39 upstream regulators of Rho-GTPases were identified of which 5 proteins had effects on both HIV-1 BaL and HIV-1 NL4.3 infection which suggests a novel role of Rho-GTPase upstream regulators in HIV- 1 infection. Early in infection, HIV-1 also encounters restriction factors such as RNA polymerase II-Associated Factor 1 (PAF1). The possible cytoplasmic or nuclear role of PAF1 in HIV-1 infection was investigated. Confocal imaging and cellular fractionation suggested that endogenous PAF1 is localised mostly in the nuclei but also in the cytoplasm. Overexpression of a murine PAF1 cytoplasm- localised mutant decreased HIV-1 infection similarly to wild-type overexpression. On the other hand, overexpression of a nuclear PAF1 construct decreased infection better than the wild-type. This suggests that PAF1 restricts HIV-1 via a nuclear function. In addition, infection time-course experiments revealed that HIV- 1 counteracts the effect of PAF1 within 30 minutes of infection. Overall, the evidence presented, suggests that PAF1 has a nuclear mechanism and that its downregulation could take place in the cytoplasm. 2 Acknowledgments Firstly, I’d like to give special thanks to Patricia Costa, Francesca Ammoscato and Aneta Kucik for their support and for our friendship during these hard four years. I couldn’t have made it without you and I’ll never forget it. I’d like to thank Deseada Camuesco, Nidia Oliveira, Ainhoa Lucia and Julieta Díaz-Delfín for their encouragement, their example and for their beautiful smiles during these years, I learnt so much from you guys and I feel gifted! I would also like to thank all the people I met at the Blizard for their support and laughs we shared, especially to Paolo Giuffrida, Margarida Rei, Renata Curciarello, Paolo Biancheri, Supatra Marsh, Filipe Almeida, Vera Cabeços, Capucine Grandjean, Tania Garcia, Dario Ceric, Simona Mazza, Valentina DiFoggia, Silvia Dibenedetto, Barbara Ricci and Dan Pennington. I’d like to thank Marjolein Snippe for her friendship, for her useful input in the FRET experiments and for counting with me for co-organising the All on Board science sessions, I really enjoyed them, thanks! I’d also like to thank Martha Wildemann for teaming up with me in the organization of the All on Board science sessions, many thanks! I’d like to thank Aysegul Tosun for her contribution to my thesis and her great enthusiasm in the lab! I’d like to thank Alex Poll, Marie Marsden and Ben Hockman for their support and love during these and previous years. I’d like to thank Alexander Ashall-Kelly and the Ashall- Kelly’s for making me feel at home during my UK years, I’ll never forget it! I’d like to thank my friends from home: Berto Arranz, Marta Hernández, Belén Jorquera, Sergio Castro, Jorge Ruiz, Rodri González, Laura Ruiz, Ana Alonso, Blanca Gil, Edu Pardo, Virginia Murialdo, Jesús Ruiz, Enrique Sánchez Bautista, Carlos Gómez and César García for showing me during years that distance does not matter. Many thanks to my girls, Irene Solís and Sara Nieto Fernández, for all the love and strength during this process. I’d like to thank my Lola for her company during the long nights in which I wrote this thesis and for her unconditional love. 3 Thank you too to the Samba da Rua family for the strength and love during the writing of this thesis! I’d also like to thank Áine McKnight for the supervision of my PhD. I’d like to thank Professor Denise Sheer for kindly accepting to be my second PhD supervisor. I’d like to thank Kelly Marno for all of her help in the lab and in the writing of my PhD thesis. I’m very grateful too for William Ogunkolade’s help in the lab. I’d also like to thank Paul Allen for his support during these tough years. I’d like to thank the BALM facility staff including Ann Wheeler, Isma Ali and Amanda Wilson for always ensuring the microscopes were ready for my experiments and for my questions. Similarly, I’d like to thank Gary Warnes for guiding me through the use of the flow cytometers and for his useful input. I’d like to thank Corina Pade and Hanna Dreja for their help at the start of my PhD. I would like to thank Cleo Bishop for all of her help during the siRNA screen. I would like to thank Eithne O’Sullivan for providing me the reagents I required and for taking care of my health and safety in the lab. I’d like to thank Jaya Rajamanie and Mary Martins for their help in the office and also the Blizard Institute’s lab managers Jeff Maskell and Chris Pelling. Bizitzari eskertu nahi diot Asier Jaio oparitu izana. Zu gabe, hau ez litzateke posible izango, mila esker, maite zaitut. Me gustaría agradecerle y dedicarle esta tesis a mi familia, que tanto me han apoyado en mis sueños, toda mi vida. Nunca lo olvidaré. A mis padres, Ana y Carlos y a mis abuelos Antonio, Antonia, Pepe y Blanca. 4 Table of contents Page Abstract 2 Acknowledgements 3 Abbreviations 10 Chapter 1. Introduction 13 1.1. Viral taxonomy 14 1.2. Retroviruses 15 1.3. Discovery and origin of Human Immunodeficiency Virus 17 1.3.1. Genetic variability and epidemiology 17 1.4. Clinical aspects of HIV infection 18 1.4.1. HIV transmission 18 1.4.2. Clinical manifestations of AIDS 19 1.5. Cell tropism 20 1.6. The HIV-1 genome 21 1.7 HIV-1 replication cycle 24 1.7.1 HIV entry and receptor engagement 26 1.7.2. Reverse transcription 28 1.7.3. Integration 31 1.7.4. Gene expression, nuclear RNA export and viral protein translation 31 1.7.5. Viral assembly, budding and maturation 33 1.8. Highly Active Antiretroviral Therapy 34 1.8.1. Nucleoside reverse transcription inhibitors (NRTIs) 35 1.8.2. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) 35 1.8.3. Fusion and co-receptor inhibitors 36 1.8.4. Integrase inhibitors 36 1.8.5. Protease inhibitors 37 1.9. Prevention strategies and vaccine attempts 37 1.9. HIV restriction 39 5 1.10.1 Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like: APOBEC3G 40 1.10.2 Tripartite-motif-containing 5α (TRIM5α) 41 1.10.3 The TRIM28 (KAP1)/SETDB1 complex 41 1.10.4 Tetherin. 41 1.10.5 SAM domain and HD domain-containing protein 1: SAMHD1 42 1.10.6 Cyclin-dependent kinase inhibitor: p21 42 1.10.7 Myxovirus resistance 2 (MX2 or MxB) protein 43 1.10.8 RNA-associated Early-stage Anti-viral Factor: REAF 43 1.10.9 The Polymerase II-Associated Factor 1 Complex 43 1.10.9.1 Discovery of the Polymerase II-Associated 44 Factor 1 Complex 1.10.9.2 The role of PAF1 in viral infection 47 Chapter 2. Materials & Methods 49 2.1 Materials: Reagents and antibodies 50 2.2. Methods 51 2.2.1 DNA preparations 51 2.2.1.1. DNA production 51 2.2.1.2. Agarose gel electrophoresis and restriction digest of DNA 52 2.2.2. Cellular fractionation 52 2.2.3 Cells 55 2.2.4 MTS cytotoxicity assays 55 2.2.5 Virus production and HIV-1 detection of infection 56 2.2.5.1 Production of single round infectious pseudovirus stocks in 293T cells 56 2.2.5.2. Production of replication-competent HIV-1 in C8166 cells 57 2.2.5.3. Detection of infection by in situ staining for β- galactosidase in TZM-bl cells 57 2.2.5.4. Detection of infection in Jurkat and PM1 cells 58 6 2.2.5.5. Determination of focus forming units by In situ immunostaining for p24 antigen 59 2.2.6. Drug-virus inhibition assays 59 2.2.6.1. Drug-virus assay in TZM-bl cells 59 2.2.6.2. Drug-virus assays in Jurkat and PM1 cells 60 2.2.6.3. Virus infection time-course. 60 2.2.7. Transfections 61 2.2.7.1 JetPEI DNA transfections of HeLa-CD4+ cells 61 2.2.7.2. Lipofectamine DNA transfections of HeLa-CD4+ cells and HIV-1 infection. 61 2.2.7.3 Transient PAF1-siRNA knockdown 62 2.2.8 siRNA screen 63 2.2.8.1.