HIV-associated neurocognitive disorders (HAND) remain among the most prominent HIV co-morbidities in the era of ART-treated HIV infection. Persistent neuroinflammation is a recognized pathogenic feature of HAND, but the reason(s) for the inflammation persisting after initiation of ART remain incompletely understood. Our recent studies demonstrated that monocytes exposed to EVs carrying HIV-1 protein Nef (Nef EVs) acquire pro- inflammatory epigenetic memory that promotes exacerbated responses to inflammatory stimuli when Nef is long gone. The mechanism of this effect is similar to “trained immunity”, a concept that stipulates that an exposure to a pathogen may not only trigger an innate immune response in myeloid cells, but also induce long-lasting metabolic and epigenetic alterations causing innate inflammatory responses to be faster and more robust. We hypothesize that this effect of Nef may also target brain myeloid cells, microglia, and possibly other brain cells, astrocytes and oligodendrocytes. If contribution of this mechanism is confirmed, that would imply that even complete elimination of the virus or virus-related pathogenic factors may not be sufficient to fully abolish neuropathology in HIV-infected individuals, as brain cells will keep their pro-inflammatory status. The following Specific Aims will be pursued to test this hypothesis. SA1. To characterize epigenetic modifications induced by HIV and Nef EVs and assess their contribution to neuroinflammation. In this Aim, we will characterize epigenetic and transcriptional effects of HIV infection and Nef EV treatment in humanized mice. Results of snATAC-seq in this model will be compared to epigenetic dataset obtained from brains of PLWH. SA2. To establish how Nef EVs induce epigenetic modifications in brain cells contributing to neuroinflammation. Here, we will establish the cell type-specific mechanisms of inflammatory modifications induced by Nef EVs. This study is a continuation of the long-term collaboration between Drs. Bukrinsky and Sviridov, who discovered the training effect of Nef on myeloid cells. The proposed project is fully consistent with the R21 mechanism, as it investigates a novel phenomenon and involves high risk high reward studies.