The overall goal of the proposed research is to come to a biochemical understanding of how a small viral protein, Tat, controls the expression of the HIV genome. The focus of the study will be on a cyclin dependent kinase, P-TEFb, that is a key factor controlling elongation by RNA polymerase II and is specifically required for Tat transactivation. HIV-1 Tat interacts with PTEFb and enhances its function at the HIV-LTR. First, human P-TEFb will be characterized by determining its subunit structure. PITALRE, the kinase subunit, and several putative cyclin subunits will be analyzed to determine if they are in complexes with active P-TEFb. Next, the requirements for efficient interaction between P-TEFb with Tat will be examined using in vitro binding studies with immobilized Tat and P-TEFb complexes and in vivo immunoprecipitation experiments. The potential requirement for protein or RNA cofactors will be addressed. Finally, a series of experiments to address the biochemical mechanism of Tat mediated enhancement of the action of P-TEFb will be performed. These will include a detailed optimization of Tat transactivation in vitro using extracts from both HeLa and Jurkat cells. The effect of Tat on the association of P-TEFb, factor 2, the SPT4/SPT5 complex, and other factors with the transcription complex will be determined. The reason that the HIV-LTR seems to be especially sensitive to PTEFb inhibitors will also be explored. In addition, a yeast system will be designed that may allow the study of Tat transactivation and the interaction of P-TEFb and Tat in vivo. The HIV epidemic is a growing national as well as international concern and the most effective treatments are those incorporating a combinatorial use of different drugs. Understanding Tat transactivation may lead to the discovery of new drugs to battle AIDS.
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| Chao, S H; Fujinaga, K; Marion, J E et al. (2000) Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J Biol Chem 275:28345-8 |
