PROJECT ABSTRACT The nature of the HIV-1 latent reservoir has recently been redefined. Our recent work has shown that a large majority of the reservoir (on average 70%) is formed near, and presumably at, the time of therapy initiation. This is a very different view of the reservoir from the historical one where the reservoir starts to be formed early after infection and forms continuously. Suppression of viral replication changes the host environment to allow certain cells, some of which are infected, to become long lived. We have also shown this is true for viral DNA even though most of it is defective. Thus, the latent reservoir is marking a set of cells that transition to a long-lived state with the initiation of therapy irrespective of their infection status. This new view of the latent reservoir begs a number of key questions that are addressed in this application. First, if the reservoir is defined in a different way does its formation have the same history? We will address this question by looking at two alternative definitions of the latent reservoir: rebound virus during therapy discontinuation, and a putative "deep" reservoir in both gut and lymph node tissue. Second, since our initial observations were made in a cohort of women, we will ask the same question about the timing of reservoir formation in men. Third, since the initiation of therapy determines when a majority of the reservoir is formed, we will examine whether the reservoir is disrupted and reforms over a period of a drug holiday followed by therapy re-initiation. Fourth, we will determine the differentiation state of the T cells harboring the subset of the reservoir that forms early prior to the initiation of therapy. Fifth, we will follow the differentiation state of cells harboring the viruses that will form the long-lived reservoir as therapy suppresses viral replication. Collectively these studies will complete our understanding of when the reservoir forms, regardless of how one defines the reservoir, in both men and women. They will also show the stability of the reservoir after reintroducing an inflammatory state that had previously largely prevented the formation of the reservoir. Finally, they will start to explore the differentiation state of the T cells that form the early reservoir and the cytokine environment present in the face of viral replication, then the differentiation state these T cells transition to as part of the long-lived reservoir during suppressive therapy. These studies will provide a conceptual framework for future new strategies to limit reservoir formation at the time of therapy initiation.