Eradicative HIV cure requires elimination of virus from all tissues, including the central nervous system (CNS), and a thorough understanding of HIV reservoirs that harbor intact and/or replication-competent virus and give rise or support rebound viremia upon treatment interruption is paramount. As long-lived CNS myeloid cells, namely microglia and resident macrophages are an HIV reservoir. While HIV enters the CNS during primary viremia and the predominant HIV variants isolated soon after infection exhibit T-cell tropism, HIV variants that infect cells with lower surface CD4 density, such as microglia, are found later during chronic infection, supported by HIV viral DNA isolated from perivascular and CNS myeloid cells and sustained HIV replication in macrophages independently of T cells in humanized myeloid-only mice. HIV from a T-cell source has also been detected in the cerebrospinal fluid (CSF) of PLWH on suppressive ART. HIV variants detected in the CSF are likely a result of a combination of independent replication of HIV compartmentalized in the CNS in CD4+ T and/or myeloid lineage cells and HIV trafficking via T cells or monocytes or as free virus from the periphery. Technical and anatomical constraints limit the comprehensive examination of (i) cellular and viral factors involved in HIV replication in the CNS and (ii) selection pressures that give rise to rebound viruses in the correct cellular context, especially in microglia. Among the reasons are limited availability of freshly isolated primary human microglia, which nonetheless rapidly lose key in vivo features, and the paucity of in vitro models that successfully recapitulate key microglial characteristics. We will use human induced pluripotent stem cell-derived microglia (iMg) differentiated from adult human fibroblasts as a tractable system to examine host and viral determinants governing rebound virus replication in CNS myeloid cells with detailed analyses that are not possible with currently available models. Two recent developments regarding rebound viruses has led to the conception of this proposal. First, in a recent study examining key viral phenotypes in patients undergoing analytical treatment interruption (ATI), our collaborator Dr. Bar found that viruses that rebounded after ATI exhibited higher IFN-1 resistance than any other viruses, including transmitted founder (TF) viruses and reservoir viruses, among viruses isolated from the blood throughout the longitudinal follow-up of patients. The origins of these uniquely IFN-1-resistant rebound viruses remain unclear. Persistent upregulation of IFN-1 signaling in the CNS of PLWH despite suppressive ART raises the possibility that selection pressures might lead to the establishment of a productive reservoir of IFN-1-resistant virus within CNS myeloid-lineage cells. Second, our preliminary data suggest that infectious molecular clones (IMCs) of some rebound viruses identified by Dr. Bar may replicate better than the IMCs of T...