Title Page Cytoplasmic CPSF6 and Cyclophilin A Modulate HIV-1 Trafficking by Zhou Zhong BS, University of Wisconsin Madison, 2012 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Medicine University of Pittsburgh 2020 Committee Membership Page UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Zhou Zhong It was defended on October 7, 2020 and approved by Alan N. Engelman, PhD, Professor; Department of Cancer Immunology and Virology, Dana- Farber Cancer Institute Paul R. Kinchington, PhD, Professor; Department of Ophthalmology and Molecular Microbiology and Genetics, University of Pittsburgh School of Medicine Nicolas Sluis-Cremer, PhD, Professor; Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine Simon C. Watkins, PhD, Distinguished Professor; Department of Cell Biology, University of Pittsburgh School of Medicine Dissertation Director: Zandrea Ambrose, PhD, Associate Professor; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine ii Copyright © by Zhou Zhong 2020 iii Abstract CYTOPLASMIC CPSF6 AND CYCLOPHILIN A MODOULATE HIV-1 TRAFFICKING Zhou Zhong, PhD University of Pittsburgh, 2020 Human immunodeficiency virus type 1 (HIV-1) capsid binds to multiple host cell proteins after entry into a cell, including cyclophilin A (CypA) and cleavage and polyadenylation specificity factor 6 (CPSF6), which is expressed predominantly in the nucleus. As CPSF6 expression was observed in the cytoplasm, we examined the effect of CPSF6 on HIV-1 capsid and nucleic acid trafficking in the cytoplasm of HeLa cells and primary macrophages. High-speed live- cell microscopy was performed with fluorescently labeled wild-type (WT) HIV-1 and capsid (CA) mutants and cells expressing labeled CPSF6. Imaging data were complimented with infectivity assays in HeLa cells, primary CD4+ T lymphocytes, and primary macrophages and in vitro binding assays with CA tubular assemblies and purified full-length CPSF6 protein and CypA. In cells, CPSF6 forms higher order complexes in the cytoplasm upon infection with WT HIV-1 but not N74D HIV-1, which does not bind to CPSF6. CPSF6 complexes associate with WT HIV-1 capsid and traffic on microtubules. Full-length CPSF6 protein and CPSF6 lacking the R/S domain (CPSF6-358) form higher order complexes that bind to and disrupt WT CA assemblies but not N74D CA assemblies in vitro. CPSF6-capsid complex trafficking can be altered by mutations in HIV-1 CA (e.g. N74D) or mutations in or truncation of the C-terminus of CPSF6 (e.g. CPSF6- 358), which is associated with decreased HIV-1 infection. In addition, disruption of HIV-1 capsid binding to CypA (e.g. cyclosporine A treatment or G89V mutation in CA) leads to increased CPSF6 binding to capsid in vivo and in vitro and altered capsid trafficking in HeLa cells and macrophages, resulting in reduced infectivity. Altered trafficking and reduced infectivity due to iv capsid-CypA perturbation can be partially restored by depletion of CPSF6 in HeLa cells but not in macrophages. Our data suggest that both CPSF6 and CypA are important for proper HIV-1 cytoplasmic capsid trafficking and infection. We propose that CypA prevents HIV-1 capsid from premature binding to cytoplasmic CPSF6. Differences in CypA cellular localization and type I interferon responses may explain cell-specific variations in HIV-1 capsid trafficking and uncoating as well as subsequent infectivity. v Table of Contents Preface ....................................................................................................................................... xxiv 1.0 Chapter 1: Introduction ......................................................................................................... 1 1.1 HIV-1 Pathogenesis and Human Health ...................................................................... 1 1.1.1 Retroviruses and HIV-1 .......................................................................................1 1.1.2 HIV-1 Pathogenesis ..............................................................................................3 1.1.3 Current Treatment...............................................................................................4 1.2 HIV-1 General Biology ................................................................................................... 7 1.2.1 HIV-1 Genome Structure ....................................................................................7 1.2.2 HIV-1 Structure ...................................................................................................8 1.2.3 HIV-1 Infection of Cells .......................................................................................9 1.2.3.1 HIV-1 Entry ............................................................................................. 9 1.2.3.2 Reverse Transcription and Capsid Uncoating .................................... 10 1.2.3.3 Nuclear Import and Integration ........................................................... 13 1.2.3.4 HIV-1 Transcription and Translation ................................................. 15 1.2.3.5 HIV-1 Packaging and Assembly ........................................................... 15 1.2.4 Evasion of Innate Restriction Factors ..............................................................16 1.2.5 Capsid Trafficking to the Nucleus ....................................................................18 1.3 Imaging HIV-1 Trafficking to the Nucleus ................................................................ 20 1.4 Specific Aims ................................................................................................................. 22 2.0 Chapter 2: HIV-1 Complexes Interact with CPSF6 in the Cytoplasm and Disruption of CPSF6 Localization Affects HIV-1 Cytoplasmic Trafficking .................... 24 vi 2.1 Project Summary .......................................................................................................... 24 2.2 Introduction .................................................................................................................. 25 2.3 Results ............................................................................................................................ 26 2.3.1 Cytoplasmic CPSF6 Traffics on Microtubules ................................................26 2.3.2 Changes in the CPSF6 RS Domain Alter WT HIV-1 Complex Trafficking 31 2.3.3 CPSF6-358 and CPSF6 Oligomerize and Disrupt Assembled WT CA .........35 2.3.4 CPSF6-358 Promotes WT HIV-1 Capsid Permeabilization after Infection of Cells ..............................................................................................................................44 2.4 Discussion ...................................................................................................................... 46 2.5 Materials and Methods ................................................................................................ 47 2.5.1 Plasmids ..............................................................................................................47 2.5.2 Cells .....................................................................................................................49 2.5.3 Viruses .................................................................................................................49 2.5.4 HIV-1 Infection Assays ......................................................................................50 2.5.5 Fluorescence Microscopy...................................................................................50 2.5.6 Imaging Quantification and Data Analysis .....................................................52 2.5.7 Protein Expression and Purification ................................................................52 2.5.8 SDS-PAGE and Western Blot Analysis ...........................................................53 2.5.9 Capsid Binding Assay ........................................................................................54 2.5.10 TEM Analysis ...................................................................................................55 2.5.11 Statistics ............................................................................................................56 2.6 Acknowledgements ....................................................................................................... 56 3.0 Chapter 3: Depletion of CPSF6 Alters HIV-1 Trafficking and Infection........................ 57 vii 3.1 Project Summary .......................................................................................................... 57 3.2 Introduction .................................................................................................................. 57 3.3 Results ............................................................................................................................ 59 3.3.1 HIV-1 Trafficking in HeLa Cells Is not Affected by CPSF6 Depletion ........59 3.3.2 CPSF6 Depletion Induces Interferon (IFN) α Production in Primary PBMCs .......................................................................................................................................60 3.3.3 Depletion of CPSF6 in Macrophages Results in Decreased HIV-1 infectivity and Altered Trafficking ..............................................................................................62 3.4 Discussion .....................................................................................................................