Mechanisms regulating HIV-1 gene expression

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DESCRIPTION (provided by applicant): The Tat protein is a viral transactivator that is activates HIV-1 gene expression by increasing the ability of RNA polymerase II to efficiently undergo transcriptional elongation. Increases in RNA polymerase II processivity can be modulated by altering the phosphorylation state of RNA polymerase II and by the association of specific elongation factors with the transcription complex. Studies indicate that Tat-activation of HIV- 1 transcription elongation is mediated by its binding to TAR RNA in conjunction to the pTEF-b kinase complex comprised of cyclin Ti and CDK9 to increase the activity of the elongation factors SPT4/SPT5. The mechanism by which the Tat/pTEF-b complex stimulates RNA polymerase IL processivity in conjunction with SPT4/SPT5 remains to be determined. In this proposal, we utilize a number of approaches in order to better understand the mechanism by which Tat increases HIV- 1 transcriptional elongation. We have used biochemical fractionation to identify two novel factors that bind to cyclin Ti and SPT5. In addition, we have found that CDK9 kinase activity is altered by changes in the cell cycle suggesting links between the regulation of the cell cycle and cellular gene expression. The changes in CDK9 kinase activity correlate with increases in the phosphorylation state of both RNA polymerase II and SPT5. Finally, we have used RINA interference, a well characterized method to interrupt specific gene function in lower species, to begin to define the in vivo transcriptional pathways involved in Tat activation. The objectives of this proposal are to elucidate the specific steps that are involved in Tat-activation of HIV- 1 gene expression. The specific aims are: (1) To characterize the function of cellular proteins JBP 1 and PP2C that are associated with SPT4/SPT5 and cyclin T1/CDK9. (2) To understand the mechanisms regulating the changes in CDK9 kinase activity during the cell cycle. (3) To determine the role of CDK9/ cyclin Ti phosphorylation of SPT5 on regulating its ability to stimulate transcriptional elongation. (4) To use RNA interference to define the transcriptional pathways that are involved in regulating Tat-activation. These studies will address the different mechanisms involved in regulating the ability of Tat to activate HIV- 1 gene expression.