Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.Viruses 2020, 12, 191 2 of 16 viral cytopathic effects, the host immune response or a second therapeutic intervention. At the same time, new infection of other cells would be controlled using antiretroviral therapy (ART) [9]. Proposed LRAs work through interfering with cellular mechanisms known to be involved in maintaining HIV-1 in a transcriptionally silent state [10]. The current commonly targeted mechanisms include (i) histone deacetylase inhibitors (HDACi) such as panobinostat, suberanilohydroxamic acid (SAHA; Vorinostat) and Trichostatin-A (TSA); (ii) bromodomain-containing protein inhibitors such as JQ1, and (iii) protein kinase C (PKC) agonists [11], which work through activation of NF-κB such as prostratin, bryostatin and ingenol. SAHA and other treatments have been unsuccessful in decreasing the size of the latent pool in vivo [9,[12][13][14][15]. Therefore, there is a need to further characterize the mechanisms that control HIV-1 transcription.The HIV-1 long terminal repeat (LTR) promoter is regulated by numerous transcription factors and chromatin-associated proteins. We have previously shown that the U3 region of HIV-1 contains a potential binding site for RUNX1 protein and that overexpression of RUNX1 and/or core binding factor-β (CBF-β) inhibits HIV-1 transcription. Conversely, suppression of endogenous RUNX1 or CBF-β with siRNA significantly increases HIV-1 transcription [16]. The benzodiazepine (BDZ) inhibitor of RUNX1/ CBF-β function, , synergizes with the HDAC inhibitor SAHA in activating HIV-1 transcription [16]. RUNX1 is one of three RUNX proteins found in humans, all of which possess a highly conserved 128 amino acid Runt DNA-binding domain at t...

The HIV-1 pandemic is a significant challenge to the field of medicine. Despite advancements in antiretroviral (ART) development, 38 million people worldwide still live with this disease without a cure. A significant barrier to the eradication of HIV-1 lies in the persistently latent pool that establishes early in the infection. The “shock and kill” strategy relies on the discovery of a latency-reversing agent (LRA) that can robustly reactivate the latent pool and not limit immune clearance. We have found that a benzodiazepine (BDZ), that is commonly prescribed for panic and anxiety disorder, to be an ideal candidate for latency reversal. The BDZ Alprazolam functions as an inhibitor of the transcription factor RUNX1, which negatively regulates HIV-1 transcription. In addition to the displacement of RUNX1 from the HIV-1 5′LTR, Alprazolam potentiates the activation of STAT5 and its recruitment to the viral promoter. The activation of STAT5 in cytotoxic T cells may enable immune activation which is independent of the IL-2 receptor. These findings have significance for the potential use of Alprazolam in a curative strategy and to addressing the neuroinflammation associated with neuroHIV-1.