PROJECT SUMMARY Current treatment for HIV infection requires strict adherence to daily antiretroviral therapy (ART). ART has greatly reduced mortality and morbidity but is not a cure. The main barrier to a cure is the pool of latently infected CD4+ T cells that carry integrated HIV provirus and are capable of reactivating and causing recrudescence of viremia if ART is stopped. A leading strategy to eliminate this persistent reservoir is termed “shock and kill” or “kick and kill” and consists of two interventions used with continued ART: first, a latency reversal agent (LRA) to reactivate latent virus and second, an approach to enhance clearance of infected cells. In this proposal, we will build upon our preliminary data indicating that activation of the non-canonical (nc) NF- κB signaling pathway with a mimetic of the second mitochondrial-derived activator of caspases (SMACm) results in systemic latency reversal in animal models. Using the highly relevant SIV/RM/ART model, we will answer three important questions regarding the biologic effects of SMACm treatment. First (Aim 1), we will determine the anatomic and cellular origin of the viremia produced during ART upon treatment with SMACm and its relationship with rebound viremia after ART interruption. In this study, we will utilize barcoded SIVmac239M to extensively sequence SIV RNA in sorted CD4+ T cell subsets from blood and tissues in comparison to plasma virus. We will also apply single cell transcriptomic methodologies to answer this question. Second (Aim 2), we test a novel “kick and kill” approach that combines latency reversal with SMACm plus venetoclax, a selective BCL-2 inhibitor, that has been shown to cause preferential apoptosis of HIV- expressing cells. We will determine whether this combined treatment reduces total, intact, and replication competent reservoirs in memory CD4+ T cells from multiple tissues and/or modulates viral recrudescence after ART interruption. Third (Aim 3), we will model the immunovirologic factors predictive of virus reactivation on ART with SMACm treatment and during ART interruption following therapeutic targeting of the ncNF-κB and BCL-2 pathways. This model will further reveal the potential of this cure approach and inform the design of future “kick and kill” studies. This work will allow us to elucidate the full potential of targeting the ncNF-κB pathway for latency reversal and to evaluate an innovative strategy to reduce virus persistence. We hope these results will contribute to a cure for HIV infection.