Targeting Astrocyte HIV-1 Proviral Reservoirs in HAND

Purpose: Although antiretroviral therapy (ART) has greatly reduced the incidence of HIV-associated dementia (HAD), nearly 50-70% of HIV-1 infected individuals develop HIV-associated neurocognitive disorders (HAND). Between 5-20% of astrocytes harbor HIV-1 provirus, and they do not actively propagate viral infection. However, it is well established that astrocytes produce viral proteins, which cause changes in astrocyte function and aggravate HAND pathogenesis. Moreover, in vivo it is difficult to distinguish latently infected astrocytes from healthy cells. Thus, there is a great need to identify latently infected astrocytes and develop strategies to target this specific population. We hypothesize that HIV-1 proviral reservoirs alter astrocyte function and gene expression patterns, which could serve as biomarkers to facilitate targeted therapy. Methods: The dual-labeled, fluorescent reporter Red/Green-HIV-1 (R/G-HIV-1) was used to visualize viral promoter (LTR) activity in primary human astrocytes. Astrocytes were spinoculated with pseudotyped R/G-HIV-1-WT. Exposed uninfected (R-/G-), astrocytes with active (R+/G+) and silent (R+/G-) LTRs were enriched using fluorescence activated cell sorting (FACS). Subsequently, these cells were used to evaluate viral protein expression, functional studies, and preliminary RNA sequencing. Results: Astrocytes with silent viral promoter are devoid of late viral proteins such as p24, indicating a functionally silent HIV-1 LTR. Vorinostat, an HDAC inhibitor, reactivated silent HIV-1 LTR in R/G-HIV-1-infected astrocytes. Preliminary data indicate that astrocytes with silent (R+/G-) and active (R+/G+) LTRs have significantly impaired glutamate clearance ability and cell proliferation compared to exposed uninfected (R-/G-) cells. Interleukin-1β (IL-1β) a HAND relevant stimuli, further reduced glutamate clearance ability of these independent populations. However, harboring the HIV-1 provirus did not alter inflammatory responses of astrocytes, such as CXCL8 and CCL2 production, either alone or in presence of IL-1β. Conclusions: Our data suggest that harboring HIV-1 provirus with either active or silent viral promoters interfered with astrocyte function and growth. Hence, we propose that identifying biomarkers for astrocytes harboring HIV provirus, and therapeutic gene editing to eliminate proviral gene expression, will improve physiological function compared to HIV-1 infected cells.