Abstract: While the latent and intact HIV reservoirs pose theoretical barriers to cure, it is also critical to understand those cells that are reactivated and/or express HIV in vivo, which contribute to immune activation and may have more potential to initiate rebound after ART interruption. However, prior studies of the “transcriptionally active reservoir” have not been able to fully characterize the heterogeneity of these cells, which vary in terms of whether they transcribe incomplete or completed and spliced HIV RNA, whether the HIV RNA is transcribed from defective or intact proviruses, and whether it is translated into HIV protein. This P01 focuses on the novel hypothesis that subsets of cells expressing different types of HIV RNA and/or protein will differ in terms of their frequency, survival or clearance rate, contribution to immune activation, cellular gene expression, differences across tissues, rebound potential, and susceptibility to new therapies aimed at HIV cure. To test these hypotheses, the three synergistic projects in this P01 will employ an array of new and cutting-edge technologies that can distinguish HIV-expressing cells based on the processivity of the HIV RNA, presence or absence of deletions or hypermutations, translation into HIV protein, and human transcriptome/proteome. Project #1 will measure the changes in blocks to HIV transcription and levels of defective or intact HIV RNA and protein over time in the blood of elite controllers and individuals who initiate ART during acute or chronic infection (aim 1), determine how they relate to immune responses and immune activation/inflammation (aim 2), and determine how differential expression of host cell genes relates to the ability of these cells to express HIV and survive in blood and lymph nodes (aim 3). In Project #2, we will measure the total burden of intact/defective proviruses and intact/defective HIV RNA across the full spectrum of different organs and tissues in vivo (aim 1), define the frequencies and phenotypes of cells expressing different types of HIV RNA transcripts across tissues (aim 2), and determine the in situ impact of HIV burden and residual transcriptional activity on host cell factors in tissue-resident lymphoid and myeloid cells (aim 3). Project #3 will determine which reservoirs of HIV- expressing cells (as well as the intact and inducible reservoir) and host factors best predict the timing of rebound in individuals who initiated ART during acute vs. chronic infection (aim 1), define the features of the rebounding virus and earliest detectable infected cells during treatment interruption (aim 2), and assess the effects of immune-based cure interventions on these HIV reservoirs (aim 3). To achieve these goals, the Bioinformatics Core will help analyze transcriptomic, proteomic, and high dimensional data, while the Administrative Core will facilitate scientific crosstalk and collaboration, coordinate allocation of resources, and track and evaluate p...