Basic Science Award

We try to understand the mechanisms of HIV transcriptional regulation as target for therapy. The viral transactivator Tat and its interacting cofactors play an important role in HIV transcription. Tat binds to an RNA structure called TAR and recruits the positive elongation factor b (P-TEFb) to HIV-1 transcripts. P-TEFb is composed of the cyclin T1 and cyclin-dependent kinase 9 (CDK9). We identified four acetylation sites in cyclin T1. Our data demonstrate that cyclin T1 acetylation activates P-TEFb through the dissociation of CDK9/cyclin T1 from inhibitory complexes 7SK RNA/Hexim1 in the basal activation of HIV LTR, but is not required for Tat transactivation. Brd4 was shown to recruits active P-TEFb to the HIV promoter in the absence of Tat. We found HDAC inhibitors treatment enhanced binding of Brd4 to cyclin T1 but did not affect the interaction of Brd4 with acetylation deficient mutant cyclin T1. Brd4 contains two bromodomains interacting with acetylated lysines. We speculate that acetylated residues in cyclin T1 bind bromodomains and regulate recruitment of active P-TEFb to the HIV LTR. To test this, we will perform coimmunoprecipitations with cyclin T1 and Brd4 bromodomains in vivo and in vitro. To assess whether acetylated cyclin T1 is recruited by Brd4 to the HIV LTR we will perform chromatin immunoprecipitation using antibodies recognizing acetylated cyclin T1 generated in Ott laboratory. Similarly, we will examine the influence of Tat against Brd4 and acetylated cyclin T1 interaction. Since Tat, cyclin T1 and TAR RNA form a complex to transactivate the HIV promoter, we will analyze bandshift assays whether cyclin T1 acetylation influences binding of P-TEFb to Tat and TAR RNA. These experiments will provide important insight into how P-TEFb regulates HIV transcription in the presence and absence of Tat for understanding of HIV transcription.