PROJECT SUMMARY Elimination of integrated, replication-competent HIV-1 proviruses from host genomes persisting despite suppressive anti-retroviral therapy (ART) is the major roadblock to a functional cure. Cells harboring these types of proviruses produce marginal levels of viral products thereby becoming refractory to immune surveillance mechanisms. This lack of detection by the immune system, in addition to its increased growth potential, due to homeostatic proliferation and clonal expansion, extend the lifespan of latently infected cells generating a persistent HIV-1 reservoir. There is enormous enthusiasm for the potential of precision therapies targeting the latent reservoir in clinical settings. To achieve this major biomedical goal, the characterization of novel basic regulatory mechanisms can pave the way to devise alternative therapeutic approaches. A large body of work has proposed that HIV-1 latency maintenance and reactivation is regulated by histone modifications. Previous studies mainly focused on the repressive roles of histone modifications involved in latency maintenance and their targeting for latency reversal. However, we have made the unexpected observation of selective H3K9me3 deposition at the provirus 3’-end during latency reactivation. This result was surprising because H3K9me3 is typically seen as a repressive histone modification, which was counterintuitive with the viral latency reversal phenotype. Despite this well-established role, H3K9me3 has also been linked to a pathway of DNA double strand breaks repair. Given these knowledge, two possible models on the function of H3K9me3 at the provirus 3’-end arise. On the viral model, it is possible that H3K9me3 deposition is used by the provirus during latency reactivation to regulate its own gene expression. On the host model, it is possible that H3K9me3 deposition is used by host cells to initiate a process of repair of damaged DNA arising as a consequence of the high levels of transcription accumulated during the exponential phase of latency reactivation. In this exploratory and developmental R21 grant application, we will test these two models using a series of genetic approaches in immortalized and primary models of latency. If successful, our studies will fill a void in our understanding of HIV-1 latency biology by describing the functions of H3K9me3 deposition at the provirus 3’-end and the enzymes implicated. In keeping with NIAID’s mission of ending the HIV-1 epidemic, our long-term objective is to leverage the basic discoveries to devise novel alternative strategies for the selective elimination of inducible HIV-1 proviruses. As such, this fundamental knowledge can be used in future studies beyond the scope of this focused grant application, to therapeutically inhibit select H3K9 methyl transferases to sensitize cells to the natural process of latency reactivation. Collectively, the proposed research will have a sustained impact in the field.