Nancie M. Archin

Expression of Latent HIV Induced by the Potent HDAC Inhibitor Suberoylanilide Hydroxamic Acid

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.

Coaxing HIV-1 from resting CD4 T cells: histone deacetylase inhibition allows latent viral expression.

Background: Histone deacetylase (HDAC), a host mediator of gene repression, inhibits HIV gene expression and virus production and may contribute to quiescence of HIV within resting CD4 T cells. Objectives: To test the ability of valproic acid (VPA), an inhibitor of HDAC in clinical use, to induce expression of HIV from resting CD4 T cells. Methods: Chromatin immunoprecipitation measured the capability of VPA to deacetylate the HIV promoter, a remodeling of chromatin linked to gene expression. The effect of VPA on resting CD4 T cell phenotype was measured by flow cytometric analysis, and its effect on de novo HIV infection of peripheral blood mononuclear cells was measured ex vivo. Outgrowth of HIV from resting CD4 T cells of aviremic, HIV-infected donors treated with highly active antiretroviral therapy was compared in limiting-dilution cultures after mitogen stimulation or exposure to VPA. Results: VPA induced acetylation at the integrated HIV proviral promoter, but CD4 cells exposed to VPA did not become activated or more permissive for de novo HIV infection. VPA induced outgrowth of HIV from the resting CD4 cells of aviremic patients at concentrations achievable in vivo as frequently as did mitogen stimulation. Conclusions: With advances in antiretroviral therapy, HIV infection might be cleared by intensive time-limited treatment coupled with practical strategies that disrupt latency without enhancing new infection. HDAC inhibitors are capable of inducing expression of quiescent provirus, without fully activating cells or enhancing de novo infection, and may be useful in future clinical protocols that seek to eradicate HIV infection.

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

BET bromodomain inhibition as a novel strategy for reactivation of HIV-1

The persistence of latent HIV‐1 remains a major challenge in therapeutic efforts to eradicate infection. We report the capacity for HIV reactivation by a selective small molecule inhibitor of BET family bromodomains, JQ1, a promising therapeutic agent with antioncogenic properties. JQ1 reactivated HIV transcription in models of latent T cell infection and latent monocyte infection. We also tested the effect of exposure to JQ1 to allow recovery of replication‐competent HIV from pools of resting CD4+ T cells isolated from HIV‐infected, ART‐treated patients. In one of three patients, JQ1 allowed recovery of virus at a frequency above unstimulated conditions. JQ1 potently suppressed T cell proliferation with minimal cytotoxic effect. Transcriptional profiling of T cells with JQ1 showed potent down‐regulation of T cell activation genes, including CD3, CD28, and CXCR4, similar to HDAC inhibitors, but JQ1 also showed potent up‐regulation of chromatin modification genes, including SIRT1, HDAC6, and multiple lysine demethylases (KDMs). Thus, JQ1 reactivates HIV‐1 while suppressing T cell activation genes and up‐regulating histone modification genes predicted to favor increased Tat activity. Thus, JQ1 may be useful in studies of potentially novel mechanisms for transcriptional control as well as in translational efforts to identify therapeutic molecules to achieve viral eradication.

Epigenetic Silencing of HIV-1 by the Histone H3 Lysine 27 Methyltransferase Enhancer of Zeste 2

ABSTRACT Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplanocin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools.

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

ABSTRACT 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.

Eradicating HIV-1 infection: seeking to clear a persistent pathogen

Effective antiretroviral therapy (ART) blunts viraemia, which enables HIV-1-infected individuals to control infection and live long, productive lives. However, HIV-1 infection remains incurable owing to the persistence of a viral reservoir that harbours integrated provirus within host cellular DNA. This latent infection is unaffected by ART and hidden from the immune system. Recent studies have focused on the development of therapies to disrupt latency. These efforts unmasked residual viral genomes and highlighted the need to enable the clearance of latently infected cells, perhaps via old and new strategies that improve the HIV-1-specific immune response. In this Review, we explore new approaches to eradicate established HIV-1 infection and avoid the burden of lifelong ART.

Immediate antiviral therapy appears to restrict resting CD4+ cell HIV-1 infection without accelerating the decay of latent infection

HIV type 1 (HIV-1) persists within resting CD4+ T cells despite antiretroviral therapy (ART). To better understand the kinetics by which resting cell infection (RCI) is established, we developed a mathematical model that accurately predicts (r = 0.65, P = 2.5 × 10−4) the initial frequency of RCI measured about 1 year postinfection, based on the time of ART initiation and the dynamic changes in viremia and CD4+ T cells. In the largest cohort of patients treated during acute seronegative HIV infection (AHI) in whom RCI has been stringently quantified, we found that early ART reduced the generation of latently infected cells. Although RCI declined after the first year of ART in most acutely infected patients, there was a striking absence of decline when initial RCI frequency was less than 0.5 per million. Notably, low-level viremia was observed more frequently as RCI increased. Together these observations suggest that (i) the degree of RCI is directly related to the availability of CD4+ T cells susceptible to HIV, whether viremia is controlled by the immune response and/or ART; and (ii) that two pools of infected resting CD4+ T cells exist, namely, less stable cells, observable in patients in whom viremia is not well controlled in early infection, and extremely stable cells that are established despite early ART. These findings reinforce and extend the concept that new approaches will be needed to eradicate HIV infection, and, in particular, highlight the need to target the extremely small but universal, long-lived latent reservoir.

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

Objectives: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. Methods:We investigated the ability of selective HDAC inhibitors to de-repress the HIV-1 LTR in both a cell line model of latency and in resting CD4+ T cells isolated from patients who were aviremic on antiretroviral therapy (ART). Results:We found that inhibition of class I HDACs increased acetylation of histones at the LTR, but that LTR chromatin was unaffected by class II HDAC inhibitors. In a latently infected cell line, inhibitors selective for class I HDACs were more efficient activators of the LTR than inhibitors that target class II HDACs. Class I HDAC inhibitors were strikingly efficient inducers of virus outgrowth from resting CD4+ T cells of aviremic patients, whereas HIV was rarely recovered from patients cells exposed to class II HDAC inhibitors. Conclusions:Further development of selective HDAC inhibitors as part of a clinical strategy to target persistent HIV infection is warranted.

Antiretroviral intensification and valproic acid lack sustained effect on residual HIV-1 viremia or resting CD4+ cell infection.

Background Human immunodeficiency virus (HIV) infection that persists despite antiretroviral therapy (ART) is a daunting problem. Given the limited evidence that resting CD4+ T cell infection (RCI) is affected by the histone deacetylase (HDAC) inhibitor valproic acid (VPA), we measured the stability of RCI and residual viremia in patients who added VPA with or without raltegravir (RAL), or enfuvirtide (ENF) with or without VPA, to standard ART. Methods Patients with plasma HIV RNA<50 c/mL added sustained-release VPA (Depakote ER®) twice daily, RAL 400 mg twice daily, or ENF 90 mcg twice daily. Change in RCI was measured by outgrowth assays. Low-level viremia was quantitated by single-copy plasma HIV RNA assay (SCA). Results In three patients on standard ART a depletion of RCI was observed after 16 weeks of VPA, but this effect waned over up to 96 weeks of further VPA. In two patients ENF added to stable ART had no effect on RCI. Simultaneous intensification with ENF and addition of VPA had no effect on RCI frequency in one patient, and resulted in a 46% decline in a second. No significant depletion of RCI (>50%) was seen in six volunteers after the addition of RAL and VPA. In 4 of the 6 patients this lack of effect might be attributed to intermittent viremia, low VPA levels, or intermittent study therapy adherence. Overall, there was no effect of the addition of RAL or ENF on low-level viremia measured by SCA. Conclusions The prospective addition of VPA and RAL, VPA and ENF, or ENF failed to progressively reduce the frequency of RCI, or ablate intermittent and low-level viremia. New approaches such as more potent HDAC inhibition, alone or in combination with intensified ART or other agents that may disrupt proviral latency must be pursued.