Persistence of the latent HIV reservoir in people living with HIV (PLWH) remains the critical barrier to an HIV cure. Numerous reservoir reduction and control studies have met with only limited success due to our profound lack of understanding of the cellular mechanisms that allow the HIV reservoir to persist during antiretroviral therapy (ART). The goals of this proposal and RFA AI-22-025 are to define the characteristics of the HIV reservoir enabling cell death resistance and to determine whether these mechanisms impact reservoir reduction strategies in PLWH. Previous studies have examined the viral reservoir from the aspect of the integrated provirus, including viral diversity, intactness, and integration site, but have not been able to directly define potential cell death resistance mechanisms that perpetuate the viral reservoir. Similarly, challenges in identifying and characterizing resting infected cells ex vivo, including rarity, heterogeneity, and absence of a defining phenotypic marker, have limited our ability to determine how the HIV reservoir is maintained under ART. To address these issues, we developed a novel single cell strategy to identify HIV+ cells via integrated proviral DNA with simultaneous epigenetic and cell surface profiling (Assayfor Transposase Accessible Chromatinsequencing with cell surface profiling and viral alignments,V-ASAPseq). Using this strategy, we have directly profiled the HIV reservoir in ART treated PLWH at the single cell level, finding extensive reservoir heterogeneity within and between individuals, but the potential for shared regulatory characteristics directly relating to cell death resistance. Here, we will apply V-ASAPseq and V-TEAseq (transcriptome, epigenome, and surface profiling) to define cell death resistance mechanisms of the HIV reservoir. Our central hypothesis is that reservoir persistence over time under ART and after immunotherapeutic challenge is associated with targetable cellular features, some of which are shared and others distinct between subpopulations of HIV+ CD4+ T cells. In Aim 1 we will determine reservoir-associated changes in cell death resistance signatures over time, between anatomical compartments and after reactivation. In Aim 2, we will determine the epigenetic and transcriptional features of HIV+ cells that drive cell death susceptibility in vitro and persistence in vivo after reservoir-targeting immunotherapies from human clinical trials. Together these studies will define targetable features of reservoir persistence and cell death both at rest, during ART, and in the context of reservoir-targeting immunotherapies with the ultimate goal of reducing the HIV reservoir.