Abstract Viral reservoir cells are an extremely small but highly durable population of HIV-1-infected CD4 T cells that persist despite treatment with highly-effective antiretroviral therapy and are responsible for viral rebound once treatment is interrupted. Understanding and characterizing the physiology of these cells will likely be critical for any effort to successfully target these cells but has turned out to be extremely difficult, due to their low fractional abundance and considerable heterogeneity in blood and tissues. Moreover, there is growing evidence suggesting that viral reservoir CD4 T cells are dynamically evolving over time, and subject to selection mechanisms that favor the long-term persistence of some reservoir cells, while eliminating others. Here, we plan to take advantage of recent progress 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 antiretroviral therapy selects for intact proviruses with features of deeper latency, likely as a result of immune selection mechanisms that preferentially eliminate proviruses more susceptible to reactivation signals, while proviruses in deeper latency persist. These studies will be conducted using samples of a unique, prospectively followed cohort of HIV-1-patients who were identified in acute infection and started antiretroviral treatment immediately after diagnosis. In Specific Aim 1, we will use novel 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. Subsequently, we will characterize the epigenetic chromatin environment within chromosomal proximity to 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 (Specific Aim 2). In Specific Aim 3, we will perform novel, functional single-cell assays to simultaneously analyze the proviral sequence, the corresponding integration sites and HIV-1 RNA expression profile of single virally infected cells from blood and tissues; this assay will allow us to individually characterize the viral gene expression pattern of single infected cells encoding for intact and defective proviruses, and enable testing the hypothesis that continuous antiretroviral therapy is associated with progressive accumulation of proviruses with deeper levels of latency and lower responsiveness to viral reactivation stimuli. Together, these studies have the potential to provide significant advances in understanding the complexity and longitudinal evolution of viral reservoir cells and may allow to identify susceptibilities and vulnerabilities of residual HIV-1-infected cells that could be therapeutically target...