University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2012-08-08 Studies on Cellular Host Factors Involved in the HIV-1 Life Cycle: A Dissertation Anna Kristina Serquiña University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Biochemistry, Biophysics, and Structural Biology Commons, Biological Factors Commons, Enzymes and Coenzymes Commons, Immune System Diseases Commons, Immunology and Infectious Disease Commons, Pharmaceutical Preparations Commons, Therapeutics Commons, Virology Commons, Virus Diseases Commons, and the Viruses Commons Repository Citation Serquiña AK. (2012). Studies on Cellular Host Factors Involved in the HIV-1 Life Cycle: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/2gzz-cy82. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/646 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Studies on cellular host factors involved in the HIV-1 life cycle Part I. Disassembly of APOBEC3G ribonucleoprotein complexes in mRNA processing bodies does not affect incorporation into HIV-1 Mentor: Tariq Rana, Ph.D. Part II. Cellular host factor UPF1 is required in an early, post-entry step of the HIV-1 life cycle Mentor: Heinrich Göttlinger, M.D. A Dissertation Presented By Anna Kristina Pilapil Serquiña Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences Worcester, Massachusetts in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 8, 2012 Biochemistry and Molecular Pharmacology "" Studies on cellular host factors involved in the HIV-1 life cycle A Dissertation Presented By Anna Kristina Pilapil Serquiña The signatures of the Dissertation Defense Committee signifies completion and approval as to style and content of the Dissertation Tariq Rana, Ph.D., Thesis Advisor Heinrich Göttlinger, M.D., Thesis Advisor Paul Clapham, Ph.D., Member of Committee Melissa Moore, Ph.D., Member of Committee Mohan Somasundaran, Ph.D., Member of Committee Ann Sheehy, Ph.D., Member of Committee (External Examiner) The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Alonzo Ross, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Biochemistry and Molecular Pharmacology August 8, 2012 """ Acknowledgements I would like to extend my heartfelt thanks to: • Heinrich Göttlinger, for adopting me into your lab and sharing your expertise in HIV-1 and for editing my introductory chapter • Tariq Rana, for the opportunity to work with you and learn from you, especially regarding how to design experiments • Alonzo Ross, chairperson of my thesis research advisory committee (TRAC) and dissertation examination committee (DEC), for the encouragement to persist and prevail against temporary roadblocks • my thesis research advisory committee (TRAC) and dissertation examination committee (DEC) members: Paul Clapham, Melissa Moore, Mohan Somasundaran, Zhuoshang Xu, and to Ann Sheehy, my external examiner. Thank you to my DEC for the encouragement, constructive comments and suggestions in improving this written dissertation. • Ken Knight, for helping me transition in the middle of graduate school • our collaborators: Melissa Moore and her team of talented postdocs and students, especially Christian Roy, for the great discussions and the helpful experiments • Siobhan O’Brien and Chia-ying Chu, for fun times inside and outside the lab • Michael Wichroski, for the mentoring you provided to this naïve graduate student • Chih-chung Lu, for helpful discussions and collaborations on APOBEC3G experiments • my colleagues at the Rana Lab: Hong Cao, Rob Nathans, Akbar Ali, G. Brett Robb, Francois Belanger, Zhonghan Li, Chaoshun Yang, Katsu Nakashima, Jie Su, Josh McCarroll, Huricha Baigude, Pam Swain, and Chris Pruitte (my surrogate aunt in UMass) • my colleagues at the Göttlinger Lab: Eric Weiss, Naomi Tsurutani, Hikaru Yamanakha, Yoshiko Usami, Sergei Popov, Elena Popova, Michio Inoue • Mark Sharkey, Maria Zapp, Paul Peters, Thomas Musich, Laura Brandano, Jim Coderre, and Ann Dauphin for helping me get started on crucial assays • my long-time classmates and compatriots: Jenny Babon (thank you for helping edit this manuscript), Rachel Madera, and Arlene Lim; also my former housemates Judith Alamares and Reina Improgo; Freidrich Cruz, Lucy Chao, Abby Guce, Rafa and Ene Carandang • my parents, Gil and Gina Serquiña, and my brothers Joachim, Dominic, and Michael, for their encouragement and support while living 12 time zones away Most especially, I would like to express my gratitude to my spouse, Cliff Lounsbury III, for your patient, loving support and for believing I could do it. "# Abstract Human Immunodeficiency Virus Type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), currently the leading cause of death from infectious diseases. Since HIV-1 co-opts the host cellular machinery, the study of cellular factors involved is a rational approach in discovering novel therapeutic targets for AIDS drug development. In this thesis, we present studies on two such proteins. APOBEC3G is from the family of cytidine deaminases known to keep endogenous retroviruses and retrotransposons at bay to maintain stability of the human genome. APOBEC3G targets Vif-deficient HIV-1 particles and renders them non- infectious, partially through deaminase-dependent hypermutation of the provirus during reverse transcription. APOBEC3G largely localizes in mRNA processing (P) bodies, cytoplasmic structures involved in RNA metabolism. Here we explore the significance of APOBEC3G localization in P bodies. We found that disrupting P bodies does not affect virion incorporation of endogenous APOBEC3G, implying that the APOBEC3G fraction in P bodies is not directly involved in the production of nascent, non-infectious particles. We also study UPF1, another host protein encapsidated by HIV-1. It is an essential protein mainly studied for its role in nonsense-mediated decay (NMD) pathway and belongs to the same helicase superfamily as MOV10, a recently identified antiviral factor. We found that UPF1 is incorporated in HIV-1 virions in a nucleocapsid-dependent manner and is required for single-cycle infectivity at an early, post-entry step of the viral life cycle. This novel function of UPF1 most likely does not involve NMD since depletion of UPF2 does not affect viral infectivity. # Table of Contents Title Page ………………………………………………………………………… i Signature Page………….………………………………………………………… ii Acknowledgements …….………………………………………………………… iii Abstract …………………...……………………………………………………… iv Table of Contents ………………………………………………………………… v List of Figures ………….………………………………………………………… viii List of Tables …………..………………………………………………………… ix List of Abbreviations …………………………………………………………….. x Chapter I: Introduction A. HIV-1/AIDS: Looking back i. An epidemic at the turn of the century ………………………….. 3 ii. HIV-1 is the causative agent of AIDS …………………………… 4 B. HIV-1/AIDs: Looking forward i. HIV/AIDS still world’s top infectious killer ……………….….… 5 ii. More accessible testing for HIV-1 …………………………….… 5 iii. Improved prognosis for patients ...……………………………..… 6 iv. Vaccine trials and prophylaxis regimens ………...………….…… 6 v. A patient is cured…………………………………………………. 9 C. HIV-1/AIDS Life Cycle i. Overview …………………………………………….….………... 9 ii. Binding to receptors and viral entry………………………………. 10 iii. Uncoating and reverse transcription ……………………………... 15 iv. Nuclear import and integration …………………………………... 20 v. Transcription of provirus and export of viral RNA………………. 22 vi. Translation of viral proteins and assembly of viral particles.….…. 23 D. Cellular host factors 25 i. Partners ……………………………………...……….….………... 28 ii. Assassins …………………………………………….….………... 28 iii. Hostages …………………………………….……….….………... 29 iv. Bystanders..……………………………………………………….. 29 E. Host factors affecting HIV-1 i. APOBEC3G: from the Old Guard of antivirals ………….…….… 30 ii. Tetherin: thwarting the viral escape ……………………………… 32 iii. TRIM5!: intruder sensor alert ..…………………………………. 35 iv. TSG101: a partner for cell break out …………………………….. 36 Chapter II : Materials and Methods A. APOBEC3G Study i. Cell culture and cell lines ………………………………………… 39 ii. Plasmids and transfections ……………………………..………… 39 iii. Virion production ………………………………………………… 40 iv. Immunostaining and confocal microscopy ….…………………… 41 v. siRNA and shRNA knockdown ……….……………………….… 41 #" vi. H9 infection and RNA extraction for miRNA microarray study … 42 B. UPF1 Study i. Transfection of proviral constructs and expression vectors ……… 43 ii. siRNA experiments …………………………………….………… 43 iii. Infectivity assays ………………………………….……………… 43 iv. Immunoblotting ………………………………………………….. 43 v. Analysis of viral particles ………………………………………... 44 vi. Analysis of virion-associated genomic RNA .………………….… 44 vii. Analysis of viral fusion ………………..……………………….… 44 viii. Analysis of post-entry events …….…………………………….…