ABSTRACT Opioid users are prime beneficiaries of cure strategies for HIV due to their medical and social vulnerabilities and low adherence to antiretroviral therapies, but the nature of the latent reservoir in people using morphine or heroin is unknown. The central hypothesis of this multi-PI proposal is that HIV DNA+ cells from opioid users show unique transcriptional and proteomic signatures that provide fundamental insight into initiation, establishment and maintenance of the latent reservoir in drug users. This hypothesis is based on our recent data in Nature characterizing HIV DNA+ CD4+ T cells without prior activation using a new sorting and sequencing strategy called FIND-Seq. We further show that specific silencing and cell survival pathways are altered in latently infected cells and identify 55 differentially expressed genes (DEGs) implicated in the underlying biology of HIV latency. Here we propose to extend these studies to tissue reservoirs from opioid users, test the functional relevance of DEGs on latency biology, and develop important genomic, proteomic and biochemical advancements of FIND-Seq. The central hypothesis will be tested in four specific aims: 1) Define the cellular mechanisms of HIV persistence and their modulation by morphine in HIV-infected T cells and microglia from Last Gift participants. We will use FIND-seq to sort and RNA sequence HIV+ cells from the spleen, gut, and brain of Last Gift participants with and without morphine to elucidate the role of opioids on latently infected T cells and microglia across different tissues. 2) Determine how DEGs and opioids regulate HIV latency in T cells and microglia. We will perform ex vivo CRISPR activation/interference experiments with isolated CD4+ T cells and microglia derived from induced pluripotent stem cells to determine the functional relevance of identified DEGs for latency establishment with and without morphine or heroin. 3) Perform a proteogenomic analysis of HIV+CD4+ T cells from the spleen of Last Gift participants. We will use DAb-seq, which allows high-coverage genome sequencing and simultaneous analysis of cell-surface protein expression on CD4+ T cells from spleens from Last Gift participants with or without morphine to determine the intactness of the provirus, its integration site, and changes in protein expression of latently infected cells induced by opioid use. 4) Simultaneously sequence the RNA, protein, and full HIV genome from HIV DNA+ cells. We will develop FIND-Seq into a multimodal assay that sorts HIV DNA+ cells on a flow cytometer and simultaneously performs genomic, transcriptomic, and proteomic analysis on recovered cells. We have assembled a highly complementary team of world experts in the biology and advanced investigation of HIV latency with cutting-edge microfluidics, CRISPR and multi-omics sequencing technologies combined with leading experts in clinical care of people living with HIV and opioid use from the Last Gift Cohort and the Johns...