Viruses have evolved intricate mechanisms for hijacking cellular resources to replicate and spread. Transcription regulation is essential for HIV to transcribe its genome and for persistent infection. The transcriptional program of HIV relies on Tat, which functions by assembling with the cellular transcription elongation factor P-TEFb on nascent viral RNAs (TAR) to activate the transition between transcription initiation and elongation. Tat captures P-TEFb from the promoter-assembled 7SK snRNP complex to activate the P-TEFb kinase. However, detailed dissection of the steps of P-TEFb release from 7SK snRNP and activation of the P-TEFb kinase when bound to TAR, is still incomplete. In addition to controlling viral transcription, Tat appears to modulate host transcription to favor the establishment of infection and affect the biology of immune system. However, the gene targets of Tat and the molecular mechanisms used remain largely unclear. This Research Plan will use interdisciplinary approaches to elucidate the mechanism by which Tat captures P-TEFb from the inactive reservoir to activate viral transcription as well as define how Tat exploits cellular complexes to impose widespread gene expression changes required for the establishment of infection. The project will be conducted at UT Southwestern Medical Center, which has state-of-the-art technologies and a very rich atmosphere of collaborators in fields other than virology such as immunology and computational biology. The work proposed builds heavily on preliminary data, a set of biochemical, genetic, virology assays that led to the identification of n enzyme recruited by Tat to 7SK snRNP to release P-TEFb and activate viral transcription. In addition, a set of genomic approaches led to the identification of direct Tat target genes, and defined how Tat alters the occupancy of Tat cofactors and the chromatin-state maps in a genome-wide scale. This preliminary data, along with a computational approach, revealed gene classes regulated by common mechanisms and revealed cellular protein complexes and chromatin-modifying enzymes as modulators of the reprogramming process. This research project will: (1) further define the mechanism of P-TEFb release from 7SK snRNP by Tat, (2) delineate how Tat targets cellular complexes to reprogram host transcription, and (3) define the role of selected Tat target genes in the pathogenesis of AIDS. Collectively, these discoveries will better elucidate the mechanism of Tat activation as well as reveal how Tat manipulates biologically relevant cellular genes to alter T cell processes.
This project will use an integrated set of genome-wide approaches to define how Tat exploits cellular resources to control both viral and host transcription. We aim to identify how Tat targets specific host complexes to better elucidate their role in the reprogramming process and the pathogenesis of AIDS. This information will aid in the better understanding of Tat in the biology of immune cells and will have broader implications for human health and disease.