The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when ART is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Our recent work suggests the inherent reservoir decay is more rapid than previously recognized. This decay is obscured due to an opposing force that results in proviral clonal expansion. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes may play a role. Overall objective: In this proposal, we dissect the drivers of reservoir contraction (Aim 1) and expansion (Aim 2). Our approach will be to perturb both forces and to study the resulting effects. We will use proviral, integration site, and RNA sequencing to understand how perturbing these forces affects the genetic make-up of proviruses, their propensity to expand, and their expression. Our approach is to perform massive deep sequencing in a few individuals rather than large sample size. We believe our intriguing results validate our deliberate decision to limit sample size to obtain deeper sequence information within each individual. With this approach, we recently provided unprecedented depth and elucidated previously unknown selection pressures. Design and Methods: In Aim 1, we isolate the role of immune clearance by measuring reservoir contraction in vivo and in vitro. We also dissect the cytotoxicity induced by HIV proteins by mutating individuals Open Reading Frames before infecting CD4 T cells with a barcoded virus. In Aim 2, we dissect the drivers of clonal expansion, including HIV-driven cell division, by using a barcoded virus. We also use longitudinal integration site analysis to compare the rate of clonal expansion as well as the “character” of the expanded proviral clones in elite controllers, acutely and chronically infected individuals on ART. The premise of our proposal is largely based on our work showing that there are two counterbalancing forces that cause (1) proviral contraction through viral cytotoxicity and immune clearance and (2) proviral expansion through clonal proliferation. The significance of our proposal includes that it may contribute to growing evidence that the reservoir is more visible than previously realized. We envision this work could lead to approaches that enhance immune clearance or target splicing to reduce reservoir size.