STUDY OF RETROVIRUS AND HOST INTERPLAY: RNA HELICASE A AND MICRORNA MODULATE VIRAL GENE EXPRESSION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shuiming Qian, B.S., M.S. * * * * * The Ohio State University 2009 Dissertation Committee: Approved by Dr. Kathleen Boris-Lawrie, Advisor Dr. Patrick Green _______________________________ Advisor Dr. Michael Lairmore Graduate Program in Molecular, Cellular, Dr. Deborah Parris and Developmental Biology ABSTRACT Retroviruses are RNA viruses that replicate through a DNA intermediate, the provirus. Provirus gene expression is dependent on the host machinery. The interplay between the virus and host post-transcriptional armamentarium is complex and the interface with the small RNA pathway has not been characterized. Retroviruses including human immunodeficiency virus type 1 (HIV-1) have evolved multiple strategies to utilize host machinery to execute intricate control of viral gene expression. As introduced in Chapter One of this dissertation, a prominent theme is that viral cis-acting RNA elements interact with cellular RNA binding proteins to modulate balanced viral post-transcriptional expression and sustain virus replication. The work in this dissertation characterized host protein interaction with a post-transcriptional control element (PCE) identified in the 5’ untranslated region (UTR) of at least eight retroviruses that facilitates efficient synthesis of retroviruses structural proteins. In addition, these studies also characterized virus-host interaction presented by the host small RNA pathway, which poses an innate cellular defense against infectious agents and retrotransposons. A growing literature shows that virus-encoded small RNAs and host encoded small RNAs play fundamental roles in animal virus replication. Another focus of the research herein was the characterization of the interaction of HIV-1 with the host small RNA pathway. The results revealed that ii virus-encoded RNA silencing suppressor activity modulates the activity of host-encoded microRNAs that can attenuate viral translation. Results of Chapter Two demonstrated for the first-time that RNA helicase A (RHA) is the cellular effector protein that operates the PCE/RNA switch. RNA mobility shift assays and RNA co-immunoprecipitation assays revealed that RHA specifically recognizes features of the redundant stem-loop structure of the PCE; the PCE/RHA interaction occurs in both the nucleus and the cytoplasm and is necessary for PCE activity. Downregulation of RHA abolishes PCE activity independently of a change in PCE mRNA stability or its cytoplasmic accumulation. Sucrose gradient analysis showed that RHA facilitates polysome accumulation of PCE-containing retroviral RNA and the cellular junD transcript. JunD is an AP-1 transcription factor and this transcript represents the first example of a cellular PCE/RHA interaction that is necessary for efficient translation. In summary, our results revealed a previously unidentified role for RHA in retrovirus and host cell translation that implicates RHA as an integrative effector of gene expression involved in the continuum of gene expression from transcription to translation. Chapter Three characterizes interplay between the host small RNA pathway and HIV-1. Experiments in plant and animal cell systems demonstrate that HIV-1 Tat regulatory protein exerts RNA silencing suppressor (RSS) activity across the plant and animal kingdoms. HIV-1 Tat and plant virus P19 RSS function similarly to suppress RNA silencing downstream of small RNA maturation. The effect of the host small RNA iii pathway was characterized by downregulation of the key enzyme of host microRNA biogenesis (Dicer), P19 expression, or by mutation in the conserved double-stranded RNA-binding domain of the Tat RSS. The outcome of the small RNA pathway on HIV-1 replication is attenuation of viral translation. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by inhibition of translation. An implication of our study is that the host small RNA pathway plays a strategic role in the viral accumulation in a newly HIV-1-infected patient. Chapter Four describes results from microRNA microarrays and functional assays that assess the interface between the host small RNA pathway the HIV-1 accessory proteins Vpr and Vif. Profiles of host microRNAs were compared between cells infected with HIV-1 or a strain deficient in vpr/vif. The outcome of this work is a microRNA microarray database that stands a resource to develop testable hypotheses about the role of microRNAs in HIV-1 biology. Protein analysis demonstrated that Vpr/Vif modulates the activity of two miRNAs that downregulate a cellular transcriptional cofactor of Tat. The results of RNA and protein analysis provide an explanation for upregulation of HIV-1 transcription during HIV-1-induced cell cycle arrest. We conclude that modulation of microRNA activity by Vpr and Vif contributes to the positive selection for conservation of vpr in HIV-1 quasispecies in infected patients. In Chapter Five, changes in microRNA profile were evaluated upon infection of human lymphocytes with an HIV-1 strain deficient in Tat RSS activity. Comparative iv analyses with HIV-1 infection demonstrated that a collection of host microRNAs are modulated by HIV-1 Tat RSS activity and indicated a generalized rather than selective effect of Tat RSS activity on host small RNA activity. Results of ribosomal profile analysis of HIV-1 transfected 293 cells determined that HIV-1 gag RNA accumulates in high molecular weight complexes that co-sediment with puromycin resistant pseudo-polysomes. Pseudo-polysomes are known sites of translational repression by microRNA. The gag transcripts redistribute to polyribosomes upon expression of viral RSS. These results document that the interface between HIV-1 and the host small RNA pathway modulates viral protein synthesis. Perspectives on the experimental results and ideas for future directions are presented in Chapter Six. In conclusion, the work in this dissertation comprehensively characterized specific viral RNA interactions with host protein RNA helicase A and the interaction of HIV-1 regulatory and accessory genes with the host small RNA pathway. Each of these interactions is important for balanced translational control of the retrovirus. v Dedicated to all members of my family, especially to my mom Yexian, my wife Xuehua, my daughter Grace and my son Aiden vi ACKNOWLEDGMENTS No single word can express my sincere gratitude for my advisor, Dr. Kathleen Boris-Lawrie, for her intellectual guidance and being supportive through this unforgettable journey. She has been the most amazing advisor and shares the knowledge she possesses and makes learning an enjoyable experience. The knowledge I gain from this experience is something that will stay with me for a lifetime and benefit me greatly. Her talent, endless encouragement and consistent support, enthusiasm about the science, unselfish training to be a good scientist, this is a wonderful opportunity to advance my career in science and have a greater understanding of a subject I am passionate about. I am grateful to my advisory committee members, Dr. Patrick Green, Dr. Michael Lairmore and Dr. Deborah Parris for their invaluable suggestions and discussions on my projects and my dissertation. I appreciate their time and continuous support. Thanks to Dr. Lianbo Yu for statistical analysis for microarray analysis and Tim Vojt for professional figure preparation. I would like to thank the past and present members of the Boris-Lawrie lab: Dr. Tiffiney Roberts Hartman, Dr. Alper Yilmaz, Dr. Marcela Hernandez, Dr. Deepali Singh, Dr. Cheryl Bolinger, Dr. Amy Hayes, Nicole Placek, Arnaz Ranji, Wei Jing, Amit Sharma vii and Xueya Liang. My special appreciation goes to Dr. Tiffiney Roberts Hartman and Dr. Alper Yilmaz for their friendship and numerous discussions on my research projects and advice on experimental designs. I also thank Dr. Cheryl Bolinger for her great discussion and sharing. Finally and most importantly, I would like to thank my parents, my wife Xuehua Zhong, my daughter Grace and my son Aiden for their love, support and encouragement. Xuehua has been my spiritual support throughout my whole career. She has been the best listener of my complaints and always refreshes my ideas. She is not only my life-mate but also soul-mate. Grace and Aiden have always been my source of energy and motivation. I have to thank my Mom for her great support in the most difficult time in my life. There are no words to say how much I love and cherish you all. viii VITA June 15, 1976 .................................................Born - Haining, P.R.China 1995-1999 ......................................................B.S. - Microbiology, Wuhan University, Wuhan, P.R.China 1999-2002 ......................................................M.S. - Molecular Genetics, Wuhan University, Wuhan, P.R.China 2003-present ..................................................Graduate Research Associate, The Department of Veterinary Biosciences The Ohio State University Columbus, Ohio PUBLICATIONS 1. Qian, S., Zhong, X., Yu, L., Ding, B. and Boris-Lawrie. K. HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1. Proceedings of the National Academy of Sciences, USA. 2009 106(2): 605-10 2. Zhong,