University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Phenotypic Characteristics of Mucosally Transmitted HIV-1 Nicholas F. Parrish University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons, and the Virology Commons Recommended Citation Parrish, Nicholas F., "Phenotypic Characteristics of Mucosally Transmitted HIV-1" (2013). Publicly Accessible Penn Dissertations. 684. https://repository.upenn.edu/edissertations/684 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/684 For more information, please contact [email protected]. Phenotypic Characteristics of Mucosally Transmitted HIV-1 Abstract Mucosal transmission accounts for the majority of new human immunodeficiency virus type 1 (HIV-1) infections and results in a genetically and phenotypically homogenous founder virus population in 60-80 percent of cases. Biological properties common to these transmitted and founder (T/F) viruses but not chronic control (CC) viruses would define ek y targets for microbicides and vaccines. To identify such properties, we tested 45 T/F and 52 CC envelope glycoproteins (Envs) from the best studied and most prevalent HIV-1 subtypes (B and C, respectively) in various pseudotype assays to determine their receptor and coreceptor interaction, tropism for primary CD4+ T cell subsets, and sensitivity to neutralizing antibodies. T/F Envs were unable to mediate entry into cells expressing low amounts of CD4, thus macrophages and other CD4low cells likely do not support their replication during mucosal transmission. In contrast, T/F Env pseudoviruses efficiently infected primary memory CD4+ T cells with a preference for the effector rather than central memory subset, as did CC pseudoviruses. There was a trend towards increased sensitivity of T/F viruses to neutralization by antibodies targeting the CD4 binding site. All T/F Envs were able to use the coreceptor CCR5 for cell entry, whereas some CC Envs used CXCR4 alone. However, one bona fide T/F virus entered and replicated very poorly in CCR5+ cells in vitro, so efficient use of CCR5 as tested is not absolutely required for transmission. To extend these studies beyond intrinsic Env functions, we characterized the Env content, infectivity, dendritic cell interaction, and interferon alpha (IFN-α) sensitivity of 27 T/F and 14 CC infectious molecular clones from subtypes B and C. T/F viruses contained more Env and were more infectious than CC viruses. T/F viruses also readily attached to DCs and were effectively transferred to CD4+ T cells. T/F viruses were more resistant to the inhibitory effect of IFN-α on virus spread than CC viruses, suggesting that selective pressure imposed by the innate immune response may in part mediate the bottleneck associated with mucosal transmission. Future work is needed to define the mechanistic basis of these phenomena in order to target them to prevent HIV-1 transmission. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Beatrice H. Hahn Subject Categories Molecular Biology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/684 PHENOTYPIC CHARACTERISTICS OF MUCOSALLY TRANSMITTED HIV-1 Nicholas F. Parrish A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation _____________________ Beatrice H. Hahn Professor of Medicine and Microbiology Graduate Group Chairperson ________________________ Daniel S. Kessler, Associate Professor, Department of Cell and Developemental Biology Dissertation Committee: Robert W. Doms, Chair and Pathologist-in-Chief, Children’s Hospital of Philadelphia James A. Hoxie, Professor of Medicine Ronald G. Collman, Professor of Medicine Scott E. Hensley, Assistant Professor, The Wistar Institute DEDICATION This dissertation is dedicated to the memory of Matthias H. Kraus, M.D., an artisan of molecular biology. He discovered ERBB3, which is the topic of over 1000 peer-reviewed publications and the target of monoclonal antibodies under clinical development as cancer therapeutics. His tutelage was a provident gift. It is solely to his credit if I lack any of the bad habits of a retrovirologist. ii ACKNOWLEDGMENT I thank my mentor Dr. Beatrice Hahn. It has been an honor to sit at the feet of such an accomplished scientist. She directed me to important problems and made sure I stayed focused on them, somehow without limiting my perceived creativity and freedom. She has encouraged me when I needed encouragement and challenged me when I needed to be challenged. The support she has provided seemed limitless. Most critically, her desire for me to improve as a scientist has been unrelenting; I sincerely hope that will continue. Dr. George Shaw has done more than anyone other than Dr. Hahn to improve the environment of my training. His perspective on HIV, especially that from “5,000 feet,” is unparalleled. I have also gained an undeserved benefactor in Dr. Robert Doms. In the same way that Dr. Shaw is the master of defining the big question, Dr. Doms is the ultimate authority on the proper manner(s) of addressing the question. I would not have withstood the challenges of the endeavor this dissertation represents without the careful sculpting of my undergraduate mentor, Dr. Bruce Levin. I am honored to share what I do of his approach and hermeneutics. I had the pleasure of receiving training and airing ideas in several external labs. The six weeks I spent at Penn in Dr. Doms’ lab prior to our move were easily the most productive of my entire four years in terms of data generation. Dr. Craig Wilen, my closest collaborator and friend, had more to do with that than I did. Dr. Nina Bhardwaj at NYU, along with Dr. Meagan O’Brien, hosted me for my first trip to dendritic cell mecca and taught me tissue culture techniques important for this work. Dr. Feng Gao allowed me to train with his group at Duke and was a critical collaborator entrusting me with many reagents his lab spent countless hours generating. Dr. Ben Greenbaum and Dr. Arnold Levine at the Institute for Advanced Studies listened to my speculation with the interest normally reserved for data, helping refine some of the hypotheses tested here. iii Committee members Dr. James Hoxie, Dr. Ronald Collman, and Dr. Scott Hensley provided exponentially more useful guidance than would be predicted based on the number of our meetings. Those to whom I am sincerely grateful for direct guidance and assistance include: Dr. Jesus Salazar-Gonzalez, a yeoman with whom I shared many late evenings in Birmingham, and Maria Salazar. Dr. Brandon Keele, who has thought more and more clearly on this topic than anyone except Dr. Hahn and Dr. Shaw. Dr. Hui Li, Dr. Fredric Bibollet-Ruche, Julie Decker, Juliet Easlick, Dr. Yingying Li, and Dr. Weimin Liu, each true professionals who patiently suffered through teaching an amateur their techniques. Fellow students in Dr. Hahn’s, Dr. Shaw’s, and Dr. Doms’ labs including Dr. Rebecca Rudicell, Dr. Rui Kong, Dr. Joshua Baalwa, Michael Lopker, Mark Stoddard, Shilpa Iyer, Dr. Jason Wojcechowskyj, Josiah Peterson, Chuka Didigu, Sesh Sundararaman, and Hannah Barbian provided helpful discussions. I especially thank my parents, who nurtured my scientific leaning from an early age and along with my in-laws continue to support my long training. I am especially proud of my dad’s blessing to pursue research and my mom’s unconditional conviction that I am great at it. Because of my passion to answer the questions raised in this work, my lifestyle over the past four years has been distinct from what I would have imagined, and with foresight and self-control, perhaps even chosen. Thus my wife Erica’s steadfast commitment to me, extending even to the same scientific pursuit, warrants gratitude and accolades for which this is not the venue. I cannot imagine a better colleague. iv ABSTRACT PHENOTYPIC CHARACTERISTICS OF MUCOSALLY TRANSMITTED HIV-1 Nicholas F. Parrish Beatrice H. Hahn Mucosal transmission accounts for the majority of new human immunodeficiency virus type 1 (HIV-1) infections and results in a genetically and phenotypically homogenous founder virus population in 60-80 percent of cases. Biological properties common to these transmitted and founder (T/F) viruses but not chronic control (CC) viruses would define key targets for microbicides and vaccines. To identify such properties, we tested 45 T/F and 52 CC envelope glycoproteins (Envs) from the best studied and most prevalent HIV-1 subtypes (B and C, respectively) in various pseudotype assays to determine their receptor and coreceptor interaction, tropism for primary CD4+ T cell subsets, and sensitivity to neutralizing antibodies. T/F Envs were unable to mediate entry into cells expressing low amounts of CD4, thus macrophages and other CD4low cells likely do not support their replication during mucosal transmission. In contrast, T/F Env pseudoviruses efficiently infected primary memory CD4+ T cells with a preference for the effector rather than central memory subset, as did CC pseudoviruses. There was a trend towards increased sensitivity of T/F viruses to neutralization by antibodies targeting the CD4 binding site. All T/F Envs were able to use the coreceptor CCR5 for cell entry, whereas some CC Envs used CXCR4 alone. However, one bona fide T/F virus entered and replicated very poorly in CCR5+ cells in vitro, so efficient use of CCR5 as tested is not absolutely required for transmission. To extend these studies beyond intrinsic Env functions, we characterized the Env content, infectivity, dendritic cell interaction, and interferon alpha (IFN-α) sensitivity of 27 T/F and 14 CC infectious molecular clones from subtypes B and C. T/F viruses contained more Env and were more infectious than CC viruses.