Regulation of human immunodeficiency virus type 1 latency by histone deacetylases

Despite highly effective antiretroviral therapies capable of suppressing plasma viremia, human immunodeficiency virus type 1 (HIV-1) persists in latent reservoirs in the millions of infected individuals worldwide. A significant contributor to viral persistence is the ability of the HIV-1 genome to stably integrate into the DNA of resting CD4+ T cells and adopt a state of latency, evading both immune detection and pharmaceutical attack. Once integrated, HIV-1 resides in the host chromatin environment where DNA is packaged around histones. Histone deacetylases (HDACs) are a family of 11 enzymes that can deacetylate histone tails, creating a repressive chromatin environment unfavorable to transcription. Enzymatic inhibition of HDACs with pan-HDAC inhibitors can reactivate latent HIV-1. However, the specific HDAC isoforms that regulate transcription from the HIV-1 5' long-terminal repeat (LTR) promoter during latency have not been completely defined. In this dissertation, I hypothesized that specific HDACs are recruited to the HIV-1 LTR during latency to maintain transcriptional repression. Using chromatin immunoprecipitation assays, I showed that the class I HDACs HDAC1, -2, and -3 are recruited to the HIV-1 LTR in the J89 cell line model of HIV-1 latency. These HDACs were highly expressed in the nuclei of resting CD4+ T cells, the primary latent HIV-1 reservoir. Targeted depletion of HDAC2 or HDAC3 using siRNA led to induction of HIV-1 expression in latency cell line models. However, simultaneous knockdown of HDAC1, -2, and -3 abolished this effect. In contrast, HDAC inhibitors that target HDAC1, -2, and -3 in tandem were potent inducers of latent HIV-1, suggesting a mechanistic difference between HDAC knockdown and enzymatic inhibition. When HDAC1, -2, or -3 were knocked down in resting CD4+ T cells isolated from aviremic, HIV-1-infected patients, we observed outgrowth of a selected number of proviral integrants. Collectively, these findings suggest that HDAC inhibitors that target a limited number of class I HDACs, specifically some combination of HDAC1, -2, and -3, may be effective antilatency therapies

Therapy for Persistent HI

Given the scope of the human immunodeficiency virus (HIV) pandemic, millions of people will be in need of chronic antiretroviral therapy (ART) for decades into the future. It is hoped that progress in prevention of HIV infection can be made, but there have been few successes in this effort thus far. At the same time, lifelong ART presents formidable problems. Therefore, while research must continue on improvements in prevention and treatment, future HIV research should now also seek to develop temporally contained therapies capable of eradicating HIV infection. This review will explore what is known about the mechanisms that restrain HIV expression and result in persistent, latent proviral infection, and what these mechanisms tell us about potential approaches towards eradication of HIV infection

Expression of latent human immunodeficiency type 1 is induced by novel and selective histone deacetylase inhibitors

A family of histone deacetylases (HDACs) mediates chromatin remodeling, and repression of gene expression. Deacetylation of histones within the HIV-1 long terminal repeat (LTR) by HDACs plays a key role in the maintenance of latency, whereas acetylation of histones about the LTR is linked to proviral expression and escape of HIV from latency. Global HDAC inhibition may adversely affect host gene expression, leading to cellular toxicities. Potent inhibitors selective for HDACs that maintain LTR repression could be ideal antilatency therapeutics

Selective HDAC Inhibition for the Disruption of Latent HIV-1 Infection

Selective histone deacetylase (HDAC) inhibitors have emerged as a potential anti-latency therapy for persistent human immunodeficiency virus type 1 (HIV-1) infection. We utilized a combination of small molecule inhibitors and short hairpin (sh)RNA-mediated gene knockdown strategies to delineate the key HDAC(s) to be targeted for selective induction of latent HIV-1 expression. Individual depletion of HDAC3 significantly induced expression from the HIV-1 promoter in the 2D10 latency cell line model. However, depletion of HDAC1 or −2 alone or in combination did not significantly induce HIV-1 expression. Co-depletion of HDAC2 and −3 resulted in a significant increase in expression from the HIV-1 promoter. Furthermore, concurrent knockdown of HDAC1, −2, and −3 resulted in a significant increase in expression from the HIV-1 promoter. Using small molecule HDAC inhibitors of differing selectivity to ablate the residual HDAC activity that remained after (sh)RNA depletion, the effect of depletion of HDAC3 was further enhanced. Enzymatic inhibition of HDAC3 with the selective small-molecule inhibitor BRD3308 activated HIV-1 transcription in the 2D10 cell line. Furthermore, ex vivo exposure to BRD3308 induced outgrowth of HIV-1 from resting CD4+ T cells isolated from antiretroviral-treated, aviremic HIV+ patients. Taken together these findings suggest that HDAC3 is an essential target to disrupt HIV-1 latency, and inhibition of HDAC2 may also contribute to the effort to purge and eradicate latent HIV-1 infection

A Limited Group of Class I Histone Deacetylases Acts To Repress Human Immunodeficiency Virus Type 1 Expression▿

Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4+ T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4+ T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4+ T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection

IC50 values of BRD3308 and SAHA for HDACs 1–9.

<p>IC50 values of BRD3308 and SAHA for HDACs 1–9.</p

Inhibition of HDAC3 induces outgrowth of HIV-1 from latently infected patient cells ex vivo.

<p>A. HIV-1 outgrowth from latently infected CD4+ T cells following overnight exposure to 15 µM BRD3308 or 335 nM SAHA. The frequency of viral outgrowth following mitogen stimulation of HDAC inhibitor exposure is expressed as infected cells per million resting CD4+ T cells (IUPM). In four patients (Pt 1, Pt 2, Pt 3, and Pt 4) exposure to BRD3308 induced a comparable frequency of outgrowth to that observed following exposure to SAHA. *IUPM for PHA for Pt.3 is obtained from three prior assays; all other assays are contemporaneous. B. PBMCs were exposed to 5, 10, 15, or 30 µM of BRD3308, and viability was measured. The results are expressed as the viability relative to the control DMSO condition. BRD3308 does not significantly affect viability of PBMCs.</p

Depletion of HDAC1, −2, and −3 significantly increases expression from the HIV-1 promoter.

<p>A. Fold change of HDAC1, −2, and −3 mRNA expression as compared to 2D10 cells transduced with the scrambled shRNA control. A significant reduction in the mRNA expression of all three HDACs was observed. B. Cell viability and proliferation as a percentage of the 2D10 cells transduced with the scrambled shRNA control. Depletion of HDAC1, −2, and −3 did not significantly affect cell viability and proliferation. C. A significant increase in the percentage of cells expressing GFP protein from the HIV-1 promoter was observed following depletion of HDAC1, −2, and −3. Furthermore, a significant increase in expression of GFP mRNA from the HIV-1 promoter was observed following depletion of HDAC1, −2, and −3. (*p<0.05).</p

Chemical inhibition of HDACs following depletion of HDAC3 significantly increases expression from the HIV-1 promoter.

<p>A. 2D10 cell that had been depleted of HDAC1, −2, or −3 or had been infected with the scrambled control lentivirus were exposed to 0.015% DMSO as the vehicle control for 24 hours. B. Chemical inhibition of HDAC1 and −2 using Mrk 12 (20 µM) does not result in a significant increase in the percentage of 2D10 cells expressing GFP following depletion of HDAC1 or −2. However, similar to depletion of HDAC3 alone, a significant increase in the percent of cells expressing GFP was observed when Mrk 12 was added to cells depleted of HDAC3. C. Chemical inhibition of HDAC1, −2 and −3 with Mrk 13 (200 nM) resulted in a significant increase in the percent of GFP positive 2D10 cells in cells depleted of HDAC2 or −3, but not HDAC1. D. A submaximal (250 nM) concentration of SAHA resulted in a significant increase in the percent of cells expressing GFP following depletion of HDAC2 and −3. E. A maximal concentration of SAHA (500 nM) resulted in a significant increase in the percent of cells expressing GFP in cells depleted of HDAC3. However, in D. or E. depletion of HDAC1 did not increase the percent of cells expressing GFP. F. Chemical inhibition of HDACs with the non-selective inhibitor TSA (25 nM) resulted in a significant increase in the percent of GFP expressing 2D10 cells in cells depleted of HDAC3, but not HDAC1 or HDAC2. G. Chemical inhibition of HDAC3, −6, and −8 with the selective HDAC inhibitor Droxinostat (2 µM) resulted in a significant increase in the percent of 2D10 cells expressing GFP in cells that had been depleted of HDAC3 but not HDAC1 or −2. (*p<0.05).</p