PROJECT SUMMARY Drug abuse and HIV infection co-exist and are difficult to study due to challenges in people living with HIV (PLWH) who abuse drugs. One of the prime areas of pathogenic interactions of HIV and drugs of abuse is the brain, which carries additional study limitations. To obtain rigorous data on the effects of HIV and cocaine on the brain, we will utilize well-studied nonhuman primate animal model, Simian immunodeficiency virus (SIV)- infected and uninfected control rhesus monkeys, in the presence or absence of cocaine. Single cell sequencing assays enable cell type-specific analysis of the role of specific cell types in both healthy brain function and disease. The overall objective of this proposal by multidisciplinary team of investigators is to reveal the single cell determinants of brain in the context of viral persistence in SIV/cART/cocaine non-human primates. Simultaneous single-cell RNA sequencing and single-cell Assay for Transposase- Accessible Chromatin sequencing (scMultiome-seq) will be carried out on cells isolated from the prefrontal cortex (PFC), striatum, and hippocampus from each animal. Data will be processed and analyzed to derive cell type-specific regulatory programs and changes in these programs that regulate neuropathogenesis in the primate brains. These studies will provide unique and valuable insights into SIV/cocaine/cART interactions in the brain while also providing crucial experimental validation of the findings in humans. Successful completion of these innovative single-cell studies and data analyses methods will generate a single cell transcriptome and epigenome atlas of cellular part list for three NIDA-relevant brain regions, PFC, striatum, and hippocampus. These results will illuminate novel insights into transcriptomic and epigenetic landscapes of CNS and how they are altered by HIV and cocaine use.