Insights into the Gene Expression Profiles of Active and Restricted Red/Green-HIV ⁺ Human Astrocytes: Implications for Shock or Lock Therapies in the Brain

Abstract

A significant number of people living with human immunodeficiency virus type 1 (HIV-1) suffer from HIV-associated neurocognitive disorders (HAND). Many previous studies investigating HIV in astrocytes as a heterogenous population have established the relevance of astrocytes to HIV-associated neuropathogenesis. However, these studies were unable to differentiate the state of infection, i.e., active or latent, or to evaluate how this affects astrocyte biology. In this study, the pseudotyped doubly labeled fluorescent reporter red/green (R/G)-HIV-1 was used to identify and enrich restricted and active populations of HIV+ astrocytes based on the viral promoter activity. Here, we report that the majority of human astrocytes restricted R/G-HIV-1 gene expression early during infection and were resistant to reactivation by vorinostat and interleukin 1β. However, actively infected astrocytes were inducible, leading to increased expression of viral proteins upon reactivation. R/G-HIV-1 infection also significantly decreased the cell proliferation and glutamate clearance ability of astrocytes, which may contribute to excitotoxicity. Moreover, transcriptome analyses to compare gene expression patterns of astrocyte harboring active versus restricted long terminal repeats (LTRs) revealed that the gene expression patterns were similar and that the active population demonstrated more widespread and robust changes. Our data suggest that harboring the HIV genome profoundly alters astrocyte biology and that strategies that keep the virus latent (e.g., block and lock) or those that reactivate the latent virus (e.g., shock and kill) would be detrimental to astrocyte function and possibly augment their contributions to HAND. IMPORTANCE More than 36 million people are living with HIV-1 worldwide, and despite antiretroviral therapy, 30 to 50% of the people living with HIV-1 suffer from mild to moderate neurocognitive disorders. HIV-1 reservoirs in the central nervous system (CNS) are challenging to address due to low penetration of antiretroviral drugs, lack of resident T cells, and permanent integration of provirus into neural cells such as microglia and astrocytes. Several studies have shown astrocyte dysfunction during HIV-1 infection. However, little is known about how HIV-1 latency affects their function. The significance of our research is in identifying that the majority of HIV+ astrocytes restrict HIV expression and were resistant to reactivation. Further, simply harboring the HIV genome profoundly altered astrocyte biology, resulting in a proinflammatory phenotype and functional changes. In this context, therapeutic strategies to reactivate or silence astrocyte HIV reservoirs, without excising proviral DNA, will likely lead to detrimental neuropathological outcomes during HIV CNS infection.

Astrocytes are HIV reservoirs in the brain: A cell type with poor HIV infectivity and replication but efficient cell‐to‐cell viral transfer

“…Normally, when LRA reactivate latently infected cells like memory CD4 + T cells, viral replication resumes. These cells become vulnerable to becoming recognized by the experienced immune system, ART (that only works in cells with active replication), and virus‐mediated cytotoxicity (Edara et al., 2020; Marban et al., 2016), which is the basis for the “shock and kill” strategy. In contrast to these latently infected T cells, viral reactivation of latently infected astrocytes occurs transiently without apoptosis or cytotoxicity.…”

“…By definition, a viral reservoir refers to long‐lived HIV‐infected cells, most of which can be quiescent and mainly localized in specific anatomical compartments, where the replication‐competent virus can persist for a longer period of time compared to the main pool of actively replicating virus (De Scheerder et al., 2019; Dufour et al., 2020; Rose et al., 2018). Apart from well‐established and accepted memory CD4 + T lymphocytes and macrophages/microglia, one cell type that still engenders debate as a potential viral reservoir are astrocytes (Barat et al., 2018; Churchill et al., 2015; Edara et al., 2020; Eugenin & Berman, 2013; Gorry et al., 2003; Ko et al., 2019; Kramer‐Hammerle et al., 2005; Li et al., 2015, 2020b; Prevedel et al., 2019).…”

Astrocyte HIV-1 Tat Differentially Modulates Behavior and Brain MMP/TIMP Balance During Short and Prolonged Induction in Transgenic Mice

“…Astrocytes are also capable of producing viral proteins such as Nef and Tat ( 67 , 68 ). Transcriptomic analysis in an in vitro model of latently infected human astrocytes found upregulated neuroinflammatory pathways including interferon signaling, death receptor signaling, and activation of pattern recognition receptors ( 69 ). Another recent study demonstrated that astrocyte-derived HIV trafficked out of CNS and was found in peripheral organs in a transgenic mouse model ( 3 ).…”

The Glutamate System as a Crucial Regulator of CNS Toxicity and Survival of HIV Reservoirs

“…However, recent clinical observations have hypothesized that an early initiation of ART is crucial to a progressive contraction of the latent HIV-1 reservoir (“shrink”). This could possibly be accomplished with simultaneous strategies that activate (“kick” or “shock”) the latent reservoir and increase the clearance of virus-infected cells (“kill”), known as a “kick-kill” or “shock-kill” strategy (Chun et al, 1999 ; Archin et al, 2012 ; Van Lint et al, 2013 ; Pace and Frater, 2019 ; Edara et al, 2020 ). Although ART suppresses viremia in HIV-1 infected individuals, infected cells used Glu to maintain their survival and latent virus reservoirs.…”