PROJECT SUMMARY The persistence of Human Immunodeficiency Virus (HIV) in long-lived, latent reservoirs remains one of the largest barriers to a functional cure. The latent reservoir consists of cells harboring replication-competent, but transcriptionally inhibited proviruses that evade immune clearance and persist in patients even after decades of antiretroviral therapy. One of the earliest strategies designed to deplete the latent reservoir was referred to as “shock and kill,” whereby latently infected cells would be induced to express the virus by treatment with latency reversing agents (LRAs) and subsequently cleared from the body. While a number of LRAs have since been described, these agents have been proven to be therapeutically untenable, at least in part due to their incomplete penetrance and notable stochasticity. The development of new LRAs for better understanding both HIV latency and transcriptional regulation, as well as for use in next-generation therapeutic strategies, is an NIH HIV/AIDS high priority research topic (NOT-OD-20-018). Several currently described LRAs work to enhance the efficiency of transcriptional elongation by directly or indirectly increasing the activity of positive transcription elongation factor b (P-TEFb). During active HIV infection, the viral Tat protein hijacks P-TEFb and recruits it to sites of nascent viral transcription. P-TEFb subsequently phosphorylates the C-terminal tail of RNA polymerase II (Pol II), licensing elongation. Recently, we described a new player in transcriptional elongation, the PAF1 complex or PAF1C. PAF1C binds to RNA Pol II at sites of transcriptional pausing, preventing P-TEFb recruitment and effectively applying a ‘parking brake’ to the transcriptional machinery. This is consistent with recent findings that PAF1C acts as a negative regulator of HIV transcription and a positive regulator of HIV latency. In our preliminary data, we report the development and initial characterization of a first-in-class small molecule inhibitor of PAF1C nucleation, termed iPAF1C. We show that iPAF1C significantly enhances the reactivation potential of several distinct LRAs in a cell line model of latency, resulting in enhanced release of RNA Pol II at integrated proviruses and enhanced transcriptional elongation. In this proposal, we seek to test the hypothesis that small molecule inhibitors of PAF1C act as effective LRAs by disruption of PAF1C nucleation and release of proximally paused RNA Pol II at the HIV promoter. First, we will test the efficacy and specificity of iPAF1C in disrupting PAF1C in J-Lat cells, using these results as benchmarks for further compound optimization (Aim 1). iPAF1C and its lead analogs will then be tested both individually and in combination with a panel of representative LRAs in a series of cell line and primary cell models of latency. Synergistic combinations will subsequently be analyzed for reactivation potential in peripheral blood mononuclear cells from people liv...