In people with HIV (PWH), infected cells persist in several tissue compartments, including brain and lymphoid organs, and can remain indefinitely during combination antiretroviral therapy (cART). This persistence is due to the ability of the virus to establish a state of latency in cells, where the proviral DNA remains transcriptionally silent for long periods. The central nervous system (CNS) is considered one of the viral reservoirs due to the blood-brain barrier, resulting in cellular compartmentalization and limited penetrability of some antiretroviral drugs. The primary cells that serve as reservoirs in the CNS compartment are microglia, macrophages, and astrocytes; however, very little is known about HIV-1 infection of these cells and the establishment of HIV-1 reservoirs in the CNS. Studies evaluating latent resting CD4+ T cells in the periphery have shown that epigenetic regulation, histone modification, transcriptional control and methylation in the 5' HIV-1 long terminal repeat (LTR), which serves as the viral promoter, facilitates viral latency. However, there remains limited understanding of how viral latency is promoted and maintained in CNS microglia and macrophages. Accordingly, this proposal aims to identify the key factors, pathways and their regulators, which determine the establishment and reactivation of latent virus in the CNS. We hypothesize that HIV-1 latency establishment, maintenance and reactivation in brain myeloid cell lineages are controlled by unique cellular factors and their regulators specific to the CNS milieu that are distinct from latently infected CD4+ T cells in the periphery. Using our recently developed in vitro multicell lineage 3D-CNS organoids with microglia from brains of HIV-1 positive donors, and infiltrating monocytes, a humanized mouse model with infected microglia, and a precise Systems Biology approach, we will define the on-off switches involved in myeloid cell reservoirs at the single cell level and HIV-1 latency in the CNS. Successful completion of this study will identify new therapeutic targets (activators and silencers) that are unique for myeloid cells in the CNS. A better understanding these factors can improve treatment of PWH and can contribute to the development of a HIV-1 cure.