The central nervous system (CNS) HIV reservoir presents a potential impediment to HIV cure, may play a role in progression of neurocognitive impairment despite treatment, and can uncommonly erupt into more severe encephalitis. This proposal focuses on this reservoir and its 'release' from antiretroviral therapy (ART)-induced inhibition of replication. Using archived and prospectively collected samples obtained in three informative clinical settings, populations in cerebrospinal fluid (CSF) and blood will be characterized by deep sequencing or single genome amplification of envelope (env) and LTR regulatory genes and their phylogenetic analysis, and the functional impact of these genes will be assessed with respect to macrophage and lymphocyte tropism and replication capacity in macrophages. These central biological features of HIV infection will define the origin and relationships of 'escaped' CSF HIV to the CNS reservoir and trace its release, expansion and intermingling with HIV derived from systemic infection. Each aim focuses on a different subject group: Aim 1 on individuals stopping ART; Aim 2 on patients with HIV encephalitis despite ART control of systemic infection; and Aim 3 on low-level asymptomatic CSF HIV escape found incidentally in cohort studies. Together these studies will provide a composite picture of the reservoir and its release, and provide critical insight into the characteristics and cellular origi of HIV that persists in the CNS in the setting of ART and remerges when ART no longer controls replication. The results will inform strategies to prevent, ameliorate or purge these reservoirs within the CNS, allowing eventual ART-free survival without HIV-related neurodegeneration.