Regulation of P-TEFb during HIV Infection

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DESCRIPTION (provided by applicant): HIV requires the function of a cellular protein, P-TEFb, that allows full length HIV transcripts to be produced. The virus synthesizes a protein, Tat, that associates with P-TEFb and brings it to the viral transcription unit through an interaction of Tat with the nascent viral transcript. P-TEFb is a cyclin dependent kinase comprised of Cdk9 and one of several possible cyclin subunits and Tat recruits only P-TEFb containing cyclinTI. A number of small compounds have been found that inhibit P-TEFb and because P-TEFb is required for the expression of most cellular genes, at high concentrations these inhibitors cause cell death. At much lower concentrations all P-TEFb inhibitors block HIV replication while having no effect on normal cellular function. The reason for this enhanced sensitivity is not known, but it is our hypothesis that P-TEFb associated molecules are responsible. To address this issue,we propose a detailed examination of the amount, subcellular location and function of P-TEFb components in commonly used cell lines and in primary human tissues relevant to HIV infection. These components include Cdk9, a newly discovered alternative form of Cdkg, cyclinT1, and 7SK, a small cellular RNA that seems to be involved in controlling the activity of P-TEFb. Biochemical studies will investigate the function of P-TEFb kinase activity and its role in transcription with an emphasis on why the expression of HIV genes is so much more sensitive to P-TEFb inhibition than normal cellular genes. Importantly, a variety of HIV infection studies will be carried out using HeLa and Jurkat cell lines and primary human monocyte derived macrophages and peripheral blood lymphocytes. In total, these studies will not only enhance our understanding of the mechanism of Tat transactivation, but will also allow us to determine how HIV infection may alter the P-TEFb environment of a cell. Finally, our results will also be useful in evaluating the potent P-TEFb inhibitor, flavopiridol, as an anti-HIV therapy.