Project Summary/Abstract Despite the advent of successful antiretroviral therapy (ART), the quest for a human immunodeficiency virus (HIV)-1 cure continues. In order to achieve total eradication, we must successfully resolve persistent viral reservoirs in various tissue compartments. HIV-associated neurocognitive disorders (HAND) remain a significant societal burden in post-ART era. In response to the RFA-MH-17-100, we propose to uncover strategies to address astrocytes as a major CNS reservoir for HIV-1. Our preliminary data show HIV-1 latency can be recapitulated in cultured human astrocytes. The lack of powerful experimental tools to evaluate latency versus reactivation has in part, been responsible for the lack of information in viral latency in the CNS. We propose to employ the powerful dual color HIV-1 reporter virus that harbors CMV-driven mCherry (red, R) and HIV-1 LTR-driven GFP (green, G). Preliminary studies using primary human astrocytes infected with RG-HIV-1 VSV-pseudotyped virions are shown. All astrocytes infected with RG- HIV-1 express the CMV-driven mCherry (red), whereas only those that have an active HIV-1 LTR will express GFP (green) and are positive for HIV-1 p24. Red-alone astrocytes (mCherry+/GFP-) represent latently infected cells amenable to easy monitoring, sorting, manipulation and mechanistic investigations. We have established this model system using primary human astrocytes, which presents a simple, convenient and powerful tool for these works. We propose that latently infected astrocytes express identifiable biomarkers, even in the absence of reactivation. And that guide RNA/Cas9 editing can excise or silence proviral gene expression to prevent reactivation and improve astrocyte physiological function. To mitigate the conundrum of distinguishing latently infected astrocytes from those that have active HIV-1 LTR function, our first goal is to identify targetable biomarkers for HIV-1 latency in human astrocytes (Aim 1). Second, we will target latently infected astrocytes without reactivation using guide RNA and Cas9 gene editing techniques to excise proviral components, rendering it silent and incapable of reactivation (Aim 2). A successful integration of Aims 1 and 2 will lead to novel therapies to cure and eradicate HIV-1 CNS reservoirs. In summary, these investigations responding to RFA-MH-17-100 will provide novel and timely insight into delineating approaches to identify and target astrocyte viral reservoirs, key CNS barriers to HIV-1/HAND cure, and eliminate them without viral reactivation.