Study of retrovirus and host interplay: RNA helicase A and microRNA modulate viral gene expression

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.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 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 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. (Abstract shortened by UMI.)...