The primary viral reservoir is an extremely small but highly durable population of HIV-1-infected CD4 T cells that persist despite treatment with highly effective antiretroviral therapy (ART) and are responsible for viral rebound once treatment is interrupted. Understanding and characterizing the physiology of these cells will likely be critical to any attempt to successfully target them, but has proven extremely difficult because of their low fractional abundance and considerable heterogeneity in blood and tissues. Moreover, there is growing evidence suggesting that CD4 T cells from the viral reservoir evolve dynamically over time and are subject to selection mechanisms that promote long-term persistence of some reservoir cells while eliminating others. Here, we plan to take advantage of recent advances in molecular single-cell and imaging analysis techniques and propose to comprehensively profile the longitudinal evolution of viral reservoir cells in blood and tissues. We hypothesize that continuous suppressive ART selects for intact proviruses with characteristics of deeper latency, likely due to immune selection mechanisms that preferentially eliminate proviruses that are more sensitive to reactivation signals, while proviruses in deeper latency persist. These studies will be conducted using samples from a unique, prospectively followed cohort of HIV-1 patients who were identified with acute infection and started ART immediately after diagnosis. In Specific Aim 1, we will use new next-generation sequencing technologies to longitudinally profile the chromosomal position of intact and defective proviruses from blood and tissues, and evaluate their microanatomical location in lymphoid tissues. Next, in Specific Aim 2, we will characterize the epigenetic chromatin environment within the chromosomal proximity of intact proviruses from blood and tissues, using a platform of next-generation sequencing assays to evaluate chromatin accessibility, inhibitory or activating histone modifications and DNA methylation. In Specific Aim 3, we will perform novel, functional single-cell assays to simultaneously analyze the proviral sequence, corresponding integration sites and HIV-1 RNA expression profile of single virally infected cells from blood and tissues; this assay allows us to individually characterize the viral gene expression pattern of single infected cells encoding intact and defective proviruses, and to test the hypothesis that continuous antiretroviral therapy is associated with progressive accumulation of proviruses with deeper levels of latency and lower response to viral reactivation stimuli. Together, these studies have the potential to make significant advances in understanding the complexity and longitudinal evolution of viral reservoir cells and may allow the identification of susceptibilities and vulnerabilities of residual HIV-1-infected cells that can be addressed therapeutically.