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Functional Control of HIV-1 Post-Transcriptional Gene Expression by Host Cell Factors

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Functional Control of HIV-1 Post-Transcriptional Gene Expression by Host Cell Factors Functional control of HIV-1 post-transcriptional gene expression by host cell factors DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amit Sharma, B.Tech. Graduate Program in Molecular Genetics The Ohio State University 2012 Dissertation Committee Dr. Kathleen Boris-Lawrie, Advisor Dr. Anita Hopper Dr. Karin Musier-Forsyth Dr. Stephen Osmani Copyright by Amit Sharma 2012 Abstract Retroviruses are etiological agents of several human and animal immunosuppressive disorders. They are associated with certain types of cancer and are useful tools for gene transfer applications. All retroviruses encode a single primary transcript that encodes a complex proteome. The RNA genome is reverse transcribed into DNA, integrated into the host genome, and uses host cell factors to transcribe, process and traffic transcripts that encode viral proteins and act as virion precursor RNA, which is packaged into the progeny virions. The functionality of retroviral RNA is governed by ribonucleoprotein (RNP) complexes formed by host RNA helicases and other RNA- binding proteins. The 5’ leader of retroviral RNA undergoes alternative inter- and intra- molecular RNA-RNA and RNA-protein interactions to complete multiple steps of the viral life cycle. Retroviruses do not encode any RNA helicases and are dependent on host enzymes and RNA chaperones. Several members of the host RNA helicase superfamily are necessary for progressive steps during the retroviral replication. RNA helicase A (RHA) interacts with the redundant structural elements in the 5’ untranslated region (UTR) of retroviral and selected cellular mRNAs and this interaction is necessary to facilitate polyribosome formation and productive protein synthesis. The research presented in this dissertation has dissected the role of ATPase-dependent helicase activity ii and ATPase-independent chaperone function of RHA in HIV-1 mRNA translation and virion RNP complex formation. The analyses of the HIV-1 RNP complexes determined that they are structurally and functionally distinct from the typical cellular mRNAs, and retain the nuclear cap-binding protein in the cytoplasm. Moreover, HIV-1 infection of lymphocytes is modulated by these interactions with the host post-transcriptional regulatory proteins. Chapter 1 introduces the current understanding and key open questions in the retrovirus RNA metabolism. This chapter focuses on two aspects of the retroviral replication cycle: (1) the post-transcriptional events of retroviral gene expression and the role of host machinery in regulating retrovirus RNA metabolism, and (2) the role of RNA helicases as important host factors in retroviral biology. This chapter presents evidence that the host-encoded RNA helicases are efficiently used by retroviruses for several steps in their replication cycle. Chapter 2 summarizes the approaches we use to define the function of host RNA helicases in viral replication. This chapter covers approaches for identification and characterization of candidate helicases and methods to define the biochemical and biophysical parameters for their specificity and functional activity. These approaches are presented in the context of cell-intrinsic and virion-associated RNA helicase activity, respectively. In order to determine the virologic scope of RNA helicase superfamily members across divergent cell lineages and viral replication cycles, the methodology and the choice of controls presented in this chapter can be effectively employed. iii Chapter 3 addresses the question if the cis-acting viral RNA element interacts with the trans-acting host factor to modulate HIV-1 translation. RHA downregulation and metabolic labeling assays determined that RHA is necessary for efficient HIV-1 RNA translation. RHA downregulation and rescue with siRNA-resistant RHA determined that the ATPase-dependent helicase activity of RHA is required and is dependent on RHA interaction with the 5’ leader. The RU5 regions of the 5’ UTR are necessary and sufficient for RHA-responsive translation. Quantitative RNA analysis determined that RHA downregulation does not affect steady state and cytoplasmic accumulation of HIV- 1 RNA, but the translational activity of the viral RNA is reduced. RHA-deficient virions are poorly infectious on primary lymphocytes and a HeLa reporter cell line. The reduction in infectivity is not attributed to changes in the levels of virion RNA, packaging of Lys-tRNA synthetase, or defective processing of reverse transcriptase. The results presented in this chapter determined that RHA is necessary for efficient cytoplasmic utilization of HIV-1 RNA as template for translation. Chapter 4 addresses the open issue of reduced infectivity of RHA-deficient HIV-1 virions. It is known that a combination of RNA helicases modulate assembly of HIV-1 non-translation/assembly RNP complexes. DHX30 and DDX24 modulate morphogenesis of HIV-1 by promoting packaging of viral genomic RNA. In this chapter, we present evidence that RHA modulates the morphogenesis of infectious HIV-1 virions. The results of membrane flotation and Gag release assays determined that RHA is necessary for membrane association and release of HIV-1 Gag in a strain-specific manner. Coimmunoprecipitation and biochemical assays determined that RHA interacts with iv HIV-1 capsid via its C-terminal domain of unknown function (DUF). Further, interaction of RHA with the 5’ UTR of viral genomic RNA is sufficient for virion incorporation of RHA. The effect of RHA downregulation and rescue with siRNA-resistant RHA determined that the ATPase-dependent helicase activity of RHA is not necessary for HIV-1 infectivity. In summary, the results presented in this chapter indicate that RHA interacts with both viral RNA and Gag structural protein to function as a chaperone that modulates morphogenesis of infectious HIV-1 virions. In the fifth chapter, the RNP complexes necessary for efficient expression of HIV-1 Rev/RRE-dependent transcripts are defined. Investigation of RNP complexes by RNA co-precipitation assays discovered that HIV-1 Rev/RRE-dependent transcripts are predominantly associated with the nuclear cap binding protein CBP80. By contrast, completely spliced viral and cellular transcripts are predominantly associated with the cytoplasmic cap binding protein, eIF4E. The results indicated that CBP80 can substitute for eIF4E to provide a tool to sustain translation during significant cellular stress, including human retrovirus infection. HIV-1 infection arrests cell cycle progression in G2/M and induces physiological stress and apoptosis, which has the potential to disrupt eIF4E-dependent translation RNP complexes of the host cell. Analysis of de novo protein synthesis demonstrated that HIV-1-induced cell cycle arrest reduces the translational activity of the cells and that viral gag mRNA translation is sustained. Exogenous expression of the HIV-1 accessory gene vpr is sufficient to suppress translation. The mechanism of translation suppression is Vpr-dependent reduced abundance of phosphorylated eIF4E and 4E-BP1. The results identified that Rev/RRE-dependent viral v RNP complexes provide a novel viral strategy to circumvent eIF4E-dependent translation initiation that sustains translation of the HIV-1 structural proteins. The final chapter summarizes the significance and future perspectives of the research presented in this dissertation. In closing, this dissertation has: (1) outlined the approaches to define the function of host RNA helicases in viral replication, (2) determined that the ATPase-dependent helicase function of RHA is necessary for HIV-1 translation, (3) determined that the RHA chaperone function promotes efficient morphogenesis of infectious HIV-1, and (4) identified a novel viral strategy to sustain HIV-1 proteins synthesis during downregulation of eIF4E-dependent translation initiation by HIV-1. In summary, the functionality of HIV-1 RNA is governed in part by RNP complexes formed by host RNA-binding proteins. Interaction with these host factors facilitate RNA:RNA and RNA:protein interactions thereby making the HIV-1 RNPs dynamic in nature. Dynamic changes in the RNP complexes are necessary for efficient viral protein synthesis, trafficking and morphogenesis of progeny virions that productively infect host lymphocytes to complete the viral life cycle. vi Dedication Dedicated to my mother and sister, without whom this would not be possible. vii Acknowledgements I would like to thank my advisor, Dr. Kathleen Boris-Lawrie, for her valuable guidance and support throughout the duration of my PhD. She is an exceptional scientist and phenomenal mentor. I would like to thank her for introducing me to the fields of retrovirology and RNA biology. I highly appreciate her efforts for teaching me the nuances of grant writing, reviewing manuscripts as well providing me the opportunity to present at various meetings. I am forever grateful to her. I would like to thank my committee members, Dr. Hopper, Dr. Musier-Forsyth Dr. Bisaro and Dr. Osmani. I appreciate their time, encouragement and valuable advice throughout this process. I would like to acknowledge Dr. Cochrane, Dr. Kvaratskhelia, Dr. Shkiriabai, Dr. Yoder and Dr. Yamaguchi for their suggestions and fruitful collaboration on various experiments presented in this dissertation. In addition, I would like to thank Dr. Lairmore, Dr. Green and Dr. Wu for their support and encouragement. I would also like to thank past and present members
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