ABSTRACT/PROJECT SUMMARY Microglia are long-lived central nervous system (CNS)-resident innate immune cells that have critical immune surveillance functions and homeostatic functions in the brain including clearance of pathogens and maintaining integrity of neuronal synapses. Microglia are targeted by HIV, have been proposed to be a reservoir for HIV persistent infection and are mediators of HIV-associated inflammation and neuropathogenesis. Importantly, microglia dysfunction is proposed to contribute to HIV associated neurodegeneration and inflammation even when HIV replication is suppressed during antiretroviral treatments. The mechanisms, direct or indirect, that drive microglia to promote inflammation during HIV infection have not been elucidated. In this study, we will utilize a primary microglia-neuron coculture model derived from pluripotent stem cells to test the hypothesis that HIV in microglia establishes persistently infected microglia that express aberrant viral RNAs, including those that retain intronic sequences, which mediate inflammasome activation to drive microglia dysfunction, CNS inflammation and neuronal injury. Our preliminary data supporting our hypothesis includes results demonstrating that primary macrophages harbor defective HIV proviruses and, despite harboring deletions and mutations that attenuate virus production, generate HIV RNAs. Furthermore, our previous work established the potential role of HIV intron containing RNA in activating inflammatory activities in macrophages and microglia. Specific questions addressed in this proposal include: 1) what is the status of proviruses in HIV infected microglia; 2) what mechanisms drive persistent expression of HIV RNA in microglia; and 3) what are the mechanisms that trigger inflammasome activation in HIV-activated macrophages? Completion of this project will provide general insights into the impact of HIV-1 persistence and expression in the context of microglia. Importantly, these studies will provide insights into mechanisms that contribute to HIV-1 CNS comorbidities which persist even with ART and could lead to new targets and strategies for treatments that will improve the lives of people living with HIV.