The latent reservoir remains the major obstacle to a cure for HIV/AIDS. Results from recent reports are consistent with the idea that the HIV reservoir cannot be accurately defined using expression of a single protein. The reported protein profiles also showed donor-dependent attributes. Better understanding is needed of what factors contribute to heterogeneity of the reservoir signatures. Our long-term goal is to develop strategies to target latently infected cells for elimination. The specific objective of the proposed research is to determine the contribution of viral tropism to molecular signatures of the latenly infected cells. Our central hypothesis is that cells latently infected with HIV will have both shared and unique molecular signatures when infected with CCR5- or CXCR4-tropic virus, tied to the phenotypic composition of infected cells. The rationale of the proposed research is to provide a molecular platform for the future development of improved strategies of reservoir targeting using antibody-based approaches, or optimization of latency reversal. We will test our central hypothesis by pursuing the following specific aims: 1) Determine how CCR5- and CXCR4-tropic infection in phenotypically different CD4+ T cells affects the characteristics of the latent reservoir; 2) Identify molecular signatures of CD4+ T cells infected with virus of different tropism; 3) Determine contribution of tropism-dependent cell markers to the performance of antibody panels to capture latently infected cells from persons with HIV. The novel aspect of our proposal is the focus on three major reservoir subsets: established in naïve cells, established in naïve cells that transitioned to memory, and established in memory cells, in the context on CXCR4- and CCR5-tropic infection. We will characterize integration frequencies and responsiveness of the reservoir to latency reversing agents (LRAs) in vitro, and identify phenotypic differences at the protein and RNA level between latently infected and uninfected cells individually within each of these subsets. The latest innovations in liquid chromatography mass spectrometry and single cell RNA sequencing will be used. Peripheral blood and tissue samples from persons with HIV will be used to determine the contribution of tropism-specific signatures of latency to the efficiency of reservoir capture ex vivo. We expect that using no more than 15 antibodies, at least 70-90% of the latent reservoir will be captured, but different antibody panels may be needed depending on the tropism of the virus infecting each individual. Our proposed multidisciplinary efforts will reveal the complete molecular signatures of cells of different phenotypic subsets infected with virus of different tropism. This research will be important for people with HIV, because it represents a significant step towards achieving the ultimate goal to reduce the latent reservoir size to the levels at which viral suppression can be sustained upon cessation...