Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2

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 flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs

Latently infected cells form the major obstacle to HIV eradication. Studies of HIV latency have been generally hindered by the lack of a robust and rapidly deployable cell model that involves primary human CD4 T lymphocytes. Latently infected cell lines have proven useful, but it is unclear how closely these proliferating cells recapitulate the conditions of viral latency in non-dividing CD4 T lymphocytes in vivo. Current primary lymphocyte models more closely reflect the in vivo state of HIV latency, but they are limited by protracted culture periods and often low cell yields. Additionally, these models are always established in a single latently infected cell type that may not reflect the heterogeneous nature of the latent reservoir. Here we describe a rapid, sensitive, and quantitative primary cell model of HIV-1 latency with replication competent proviruses and multiple reporters to enhance the flexibility of the system. In this model, post-integration HIV-1 latency can be established in all populations of CD4 T cells, and reactivation of latent provirus assessed within 7 days. The kinetics and magnitude of reactivation were evaluated after stimulation with various cytokines, small molecules, and T-cell receptor agonists. Reactivation of latent HIV proviruses was readily detected in the presence of strong activators of NF-κB. Latently infected transitional memory CD4 T cells proved more responsive to these T-cell activators than latently infected central memory cells. These findings reveal potentially important biological differences within the latently infected pool of memory CD4 T cells and describe a flexible primary CD4 T-cell system to evaluate novel antagonists of HIV latency.     

The role of integration and clonal expansion in HIV infection: live long and prosper

Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.     

Paucity of HIV DNA methylation in latently infected, resting CD4+ T cells from infected individuals receiving antiretroviral therapy

Maintenance of HIV latency in vitro has been linked to methylation of HIV DNA. However, examinations of the degree of methylation of HIV DNA in the latently infected, resting CD4(+) T cells of infected individuals receiving antiretroviral therapy have been limited. Here, we show that methylation of the HIV 5' long terminal repeat (LTR) in the latent viral reservoir of HIV-infected aviremic individuals receiving therapy is rare, suggesting that other mechanisms are likely involved in the persistence of viral latency.     

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.     

Effect of latent human immunodeficiency virus infection on cell surface phenotype.

Highly active antiretroviral therapy has succeeded in many cases in suppressing virus production in patients infected with human immunodeficiency virus (HIV); however, once treatment is discontinued, virus replication is rekindled. One reservoir capable of harboring HIV in a latent state and igniting renewed infection once therapy is terminated is a resting T cell. Due to the sparsity of T cells latently infected with HIV in vivo, it has been difficult to study viral and cellular interactions during latency. The SCID-hu (Thy/Liv) mouse model of HIV latency, however, provides high percentages of latently infected cells, allowing a detailed analysis of phenotype. Herein we show that latently infected cells appear phenotypically normal. Following cellular stimulation, the virus completes its life cycle and induces phenotypic changes, such as CD4 and major histocompatibility complex class I down-regulation, in the infected cell. In addition, HIV expression following activation did not correlate with expression of the cellular activation marker CD25. The apparently normal phenotype and lack of HIV expression in latently infected cells could prevent recognition by the immune response and contribute to the long-lived nature of this reservoir.     

A stochastic model of latently infected cell reactivation and viral blip generation in treated HIV patients

Motivated by viral persistence in HIV+ patients on long-term anti-retroviral treatment (ART), we present a stochastic model of HIV viral dynamics in the blood stream. We consider the hypothesis that the residual viremia in patients on ART can be explained principally by the activation of cells latently infected by HIV before the initiation of ART and that viral blips (clinically-observed short periods of detectable viral load) represent large deviations from the mean. We model the system as a continuous-time, multi-type branching process. Deriving equations for the probability generating function we use a novel numerical approach to extract the probability distributions for latent reservoir sizes and viral loads. We find that latent reservoir extinction-time distributions underscore the importance of considering reservoir dynamics beyond simply the half-life. We calculate blip amplitudes and frequencies by computing complete viral load probability distributions, and study the duration of viral blips via direct numerical simulation. We find that our model qualitatively reproduces short small-amplitude blips detected in clinical studies of treated HIV infection. Stochastic models of this type provide insight into treatment-outcome variability that cannot be found from deterministic models.     

Establishment and maintenance of HIV latency: model systems and opportunities for intervention

HAART has succeeded in reducing morbidity and mortality rates in patients infected with HIV. However, a small amount of replication-competent HIV can persist during HAART, allowing the virus to re-emerge if therapy is ceased. One significant source of this persistent virus is a pool of long-lived, latently infected CD4(+) T cells. This article outlines what is known about how this reservoir is established and maintained, and describes the model systems that have provided insights into the molecular mechanisms governing HIV latency. The therapeutic approaches for eliminating latent cells that have been attempted are also discussed, including how improvements in understanding of these persistent HIV reservoirs are being used to develop enhanced methods for their depletion.     

HIV latency in the humanized BLT mouse

Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4(+) T cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevant in vivo models that can be used to test purging strategies. Here, we show that the humanized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivo latency analysis and also as an in vivo system for the study of latency. We demonstrate that over 2% of human cells recovered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIV ex vivo from these latently infected cells, indicating that this model can be used as a source of primary cells for testing latency activators. Finally, we show activation-inducible virus is still present following suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess the in vivo efficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistent in vivo HIV reservoirs.