Viruses have evolved intricate mechanisms for hijacking cellular resources to replicate and spread. Over the past two decades, great progress has been made in identifying hundreds of cellular factors involved in HIV transcription and describing their critical importance in AIDS pathogenesis. However, there is currently a dearth of information regarding how these protein complexes communicate to sustain active transcription, and how their deregulation leads to the establishment of latency. The identification of new regulatory factors, their assembly and function will provide not only mechanistic insights but it will elucidate novel candidate targets for HIV eradication strategies. This research project aims at further investigating the role of Tat and novel host co-factors in HIV transcription and their role in promoting viral latency. Tat activates the transition into elongation by transferring P-TEF from the inhibitory, promoter-bound 7SK snRNP to the TAR element on nascent viral pre-mRNAs. In this model, Tat recruits the PPM1G phosphatase to the promoter-bound 7SK snRNP to dephosphorylate the T loop of the P-TEFb kinase thereby locally releasing P-TEFb to activate transcription elongation. However, detailed dissection of the steps of: (i) 7SK snRNP recruitment to the HIV promoter to restrict transcription elongation, (ii) assembly of the Tat-PPM1G-7SK complex to release P-TEFb, and (iii) the role of PPM1G in sustaining viral transcription, is still incomplete. This research project will use a wide- range of approaches to elucidate the mechanism by which the 7SK snRNP is recruited to the HIV promoter and how Tat assembles with PPM1G to capture P-TEFb from the snRNP to activate viral transcription. The work proposed builds on a recent publication and a novel set of preliminary data that identified: (i) KAP1/TRIM28 as a possible factor that recruits the 7SK snRNP to the HIV promoter to restrict proviral transcription, and (ii) novel features of the assembly of PPM1G into the 7SK snRNP (to release P-TEFb and activate HIV transcription). Given that these Tat co-factors restrict proviral transcription, they emerge as key candidates for rationale HIV eradication strategies. The project will be conducted at U.T. Southwestern Medical Center, which has state-of-the-art technologies and a very rich atmosphere of collaborators in fields other than virology. An ongoing collaboration with Dr V. Planelles (University of Utah) will be critical to provide a mechanistic understanding of latency in primary cells. Collectively, these discoveries will elucidate the role of these cellular factors in proviral transcription and latency, and will provid novel opportunities to reactivate latent HIV.
This project will combine a wide-range of approaches to further understand the process of HIV transcription and latency. We aim to precisely delineate how Tat exploits novel host co-factors for HIV transcription activation and how deregulation of these processes leads to transcriptional silencing and entry into latency, a barrier for HIV eradication. This information will aid in an incremental understanding of Tat activation and will provide novel therapeutic opportunities towards efforts for a functional cure.
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