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Vpu Mediated Enhancement of Human Immunodeficiency Virus Pathogenesis: the Role of Conserved and Unique Domains in Protein Function

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Vpu Mediated Enhancement of Human Immunodeficiency Virus Pathogenesis: the Role of Conserved and Unique Domains in Protein Function VPU MEDIATED ENHANCEMENT OF HUMAN IMMUNODEFICIENCY VIRUS PATHOGENESIS: THE ROLE OF CONSERVED AND UNIQUE DOMAINS IN PROTEIN FUNCTION BY AUTUMN JOY RUIZ Submitted to the graduate degree program in Anatomy and Cell Biology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy ________________________ Edward B. Stephens, Ph.D. Chairperson ________________________ Nancy Berman, Ph.D. ________________________ Charlotte Vines, Ph.D. ________________________ Michael Werle, Ph.D. ________________________ Thomas Yankee, Ph.D. Date Defended: November 22, 2010 The Dissertation Committee for Autumn Joy Ruiz certifies that this is the approved version of the following dissertation: VPU MEDIATED ENHANCEMENT OF HUMAN IMMUNODEFICIENCY VIRUS PATHOGENESIS: THE ROLE OF CONSERVED AND UNIQUE DOMAINS IN PROTEIN FUNCTION ______________________________ Edward B. Stephens, Ph.D Chairperson Date approved: December 13, 2010 ii II. Abstract The work in this dissertation examined the biological characteristics of different HIV-1 Vpu subtypes, with an emphasis on subtypes B and C, and the potential impact of these proteins on SHIV pathogenesis. Different Vpu subtypes exhibited distinct biological properties that potentially could affect HIV-1 pathogenesis and/or transmission efficiency, including intracellular localization, efficiency in down-modulating CD4 surface expression, and the ability to enhance virion release in HeLa cells. We show for the first time that the subtype B and C Vpu proteins partitioned into detergent resistant membranes (DRMs), a property characteristic of lipid raft association. We also identified two mutants, IVV19-21AAA and W22A, that prevented this association. Additionally, we found a correlation between the ability of Vpu to stably associate with DRMs and the ability to enhance virion release in HeLa cells. Analysis of different Vpu proteins from clinical isolates identified a membrane proximal tyrosine motif (YXX ) that is highly conserved among all Vpu subtypes and an overlapping dileucine motif ([D/E]xxxL[L/I]) that is conserved among subtype C Vpu proteins. Substitution of the tyrosine residue in the YXX motif with an alanine (Y35A) significantly inhibited SHIV replication while substitution of the primary leucine residue with a glycine (L39G) in the overlapping [D/E]xxxL[L/I] motif significantly increased the amount of virus released from C8166 cells and the mean number of viral particles per cell compared to cells inoculated with the parental SHIVSCVpu. Recently, the enhanced virion release function of Vpu has been attributed to the antagonism of bone marrow stromal antigen 2 (BST-2) protein. This has been shown to involve the transmembrane domains (TMD) of both proteins. Our results indicate that the length of the BST-2 TMD is more important than the primary sequence both for the function and sensitivity of the protein. Additionally, we showed that the BST-2 protein expressed in pig-tailed macaques is not iii antagonized by HIV-1 Vpu, but rather by SIV Nef, thus indicating a species-specific basis for this antagonism. Based on these results we continued our analyses by examining the biological characteristics of SHIV expressing either a subtype B (SHIVKU-1bMC33) or C (SHIVSCVpu) Vpu + protein. SHIVSCVpu caused a more gradual rate of CD4 T cell loss and lower peak viral loads in infected pig-tailed macaques compared to macaques inoculated with SHIVKU-1bMC33. The identification of the TMD and the putative sorting signals proximal to the membrane as key determinants in Vpu-mediated enhanced virion release prompted the hypothesis that these two regions may dictate these differences in pathogenesis. Therefore, we constructed chimeric Vpu proteins in which the N-terminus/TMD regions of the subtype B and C Vpu proteins were exchanged (VpuBC and VpuCB). Inoculation of pig-tailed macaques with SHIVVpuCB resulted + in a more gradual loss of circulating CD4 T cells compared to SHIVVpuBC-inoculated macaques, but more rapid than resulted in macaques inoculated with parental SHIVSCVpu. Since both of these proteins down-modulated CD4 surface expression similar to the unmodified VpuSCEGFP1 protein, our results indicate that the differences observed in CD4 surface down-modulation in vitro are most likely not physiologically relevant. Finally, as pig-tailed macaques express a BST-2 protein that is not affected by the HIV-1 Vpu protein, our results suggest that the enhanced virion release function of different Vpu subtypes as observed during an intravenous inoculation may be dependent upon a different factor(s). The work presented here demonstrates a clear potential for differential signaling and functional efficiency among all HIV-1 Vpu subtypes with the ability to modify pathogenesis. Additionally, our analyses identified the TMD and membrane proximal regions as crucial components to Vpu enhancement of pathogenesis providing novel information essential for anti-retroviral therapeutic development. iv III. Acknowledgments The graduate career of a student is an exciting, challenging and humbling experience. For the past five years I have been surrounded by great minds and enormous support that have made my experience at KUMC remarkable and I would like to take this opportunity to thank those individuals who have been a part of it. First, I would like to thank my mentor, Dr. Edward B. Stephens, for all of your help and guidance. You have always encouraged me to work-hard and continually challenge myself and I hope you view this dissertation as a positive reflection of your mentorship like I do. I would also like to thank Drs. Nancy Berman, Charlotte Vines, Michael Werle, and Tom Yankee for all of their support and the time and effort they spent serving on my comprehensive exam and dissertation committees. I would also like to thank all of the members of the Microbiology, Molecular Genetics, and Immunology department as well as the Anatomy and Cell Biology department. I would like to send a special thanks to Dr. Doug Wright for his continued support and guidance throughout the years. Thank you to the members of the Stephens’ lab, Sarah Hill and Kim Schmitt. Sarah, words cannot express my gratitude. Your intellect astounds me and working alongside of you was a privilege. Thank you for always lending a helping hand when I discovered new experiments that “hate me” and for all of the random discussions that helped me to think through a problem or discover new directions. I owe a major part of my growth as a scientist to you and I thank you from the bottom of my heart. Kim, you are an incredible friend and an amazing scientist and I would never have made it through this without you. You have been through so much these past five years and I am amazed by your perseverance and positive outlook. I thank you for all of your help and for the random questions and discussions that helped me put together this body of work. I especially v thank you for all of the times you helped me find my way when I got lost, particularly when it included shopping, movies and margaritas. You are an incredibly wonderful and gifted person and I am proud to call you my friend and my coworker. I would also like to extend my gratitude to the former members of the Stephens’ lab: Erik Pacyniak, Jean-Marie Miller, Dr. Dave Hout, Fenglan Jia, Zhengian Liu, and Istvan Adany for all of their contributions. Also, thank you to our collaborators in Dr. John Guatelli’s lab at the University of California, San Diego, for all of their work and insight. To my friends and family, I apologize that I cannot single all of you out, but please know that you mean the world to me and that I couldn’t have done this without you. I love you all and I hope to make you proud. To my mom, Echo, thank you for being the single greatest support system anyone could ever ask for. As some would say I am a “character” and I do some crazy things, but your love and support have never faltered and for that I am eternally grateful. I owe all that I am and everything I have had the opportunity to do in my life to you. You are one of the bravest, most kind-hearted, intelligent people I know and I would not have made it through this experience without you. I will never be able to thank you enough for everything you do for me, but I hope to make you as proud of me as I am to call you mom. To my brother, Matt, you are one of the most intelligent, witty and generous persons I have ever met. You have always been an inspiration to me to continually challenge myself and keep moving forward. I admire your courage, your eagerness for learning and your limitless ability to question all that you encounter. I love you very much and I thank you for being who you are, for always being able to make me laugh and for inspiring me to be a better person. vi To my dad, never does a day go by that I don’t think of you. You were one of the most kind, humble and joyful people I have ever known. You supported me through everything even if it was crazy and I know you have been with me through this whole experience. You taught me to work hard, enjoy life, and help others as often as I could and I will continue to do just that. I thank you for being a wonderful father and for all of the support and love I know you still send. To Ryan, I love you with everything that I have and all that I am. Your love and support these past few years have been immeasurable and I will forever be indebted to this experience for bringing us together.
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