Opioid driven exacerbations of neuropathological events and alterations in HIV transcription contributing to HIV associated CNS dysfunction are well-reported. Despite years of continuous suppressive antiretroviral therapy (ART), latent HIV persists and finds sanctuary in many of the same brain regions involved in opioid use disorder (OUD) suggesting interactions between HIV and opioids in brain cells. However, there is a sizeable gap in our knowledge on how OUD impacts cellular responses and viral persistence in HIV-infected brain on ART in humans or relevant model organsims. This proposal seeks to generate topographical data sets and evidence at single cell resolution across the hippocampus and prefrontal cortex (PFC), two brain regions known for predilection for HIV persistence and OUD in non-human primate (NHP) and in post-mortem human brain. These data will provide an unprecedented cellular landscape of multiple modalities that can be harnessed to develop strategies to limit viral persistence and restore and retain optimal brain health in people living with HIV. In our published and preliminary work we have developed innovative single-cell approaches: (A) Single-cell isoform RNA sequencing (ScISOr-Seq), which enables single-cell long-read RNA sequencing of polyadenylated RNAs across thousands of single cells; (B) Slide-isoform sequencing (Sl-ISO-Seq) to spatially locate isoforms in brain slices and (C) a single-cell platform that identifies HIV sequences at single cell level (ScHIV-Seq). In concert these novel sequencing and computational methods, along with scATAC-Seq for chromatin accessibility, will permit the mapping of cellular gene expression, open chromatin regions, isoforms and the detection of HIV across single- cells of hippocampus and PFC. Recent literature supports the presence of HIV in the brain and more specifically in microglia and astrocytes present within the hippocampus and PFC. Importantly, these brain regions are also involved in associative learning processes for OUD. Moreover, our prior studies in rodent hippocampus have laid the groundwork for the proposed studies by establishing the regional and cell-specific distributions of opioid peptides and receptors as well as related signaling molecules, and how these distributions are impacted by sex, stress and opioid-associated learning. In further preliminary studies, we conduct opioid receptor mapping, brain spatial transcriptomics, NHP cognitive behavioral assessment and pharmacological profiling of current ART regimens in tissues. These approaches will provide a comprehensive regional landscape to support our single cell specific phenotypes. We propose an overarching hypothesis that: (i) our new integrated single-cell methods will map single-cell and cell-type specific human and NHP transcriptome and epigenome signatures in the hippocampus and PFC of S/HIV in NHPs and post-mortem human brain; (ii) chronic opioid exposure adds a distinguishable signature to S/HIV infectio...