Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection.

The latent reservoir for HIV-1 in resting memory CD4+ T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4+ T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4+ T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4+ T cells. Establishment of latent HIV-1 infection in CD4+ T could be inhibited by viral-specific CD8+ T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines.

Unique characteristics of histone deacetylase inhibitors in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells.

OBJECTIVES: The latent reservoir for HIV-1 in resting memory CD4+ T cells is a major barrier to eradication. In vitro models involving transformed cell lines have been used to search for small molecules that reactivate latent HIV-1. Histone deacetylase (HDAC) inhibitors can reverse HIV-1 latent infection. Most studies on HDAC inhibitors have been performed in cell line models that differ in important aspects from the resting CD4+ T cells that harbour latent HIV-1 in vivo. Therefore, we evaluated the potency and kinetics of HDAC inhibitors in a primary cell model of HIV-1 latency that involves resting CD4+ T cells. METHODS: A green fluorescent protein (GFP)-expressing reporter virus NL4-3-Δ6-drGFP was used to generate latent infection in Bcl-2-transduced primary CD4+ T cells. Seventeen HDAC inhibitors were tested in this primary cell model. The effects of these HDAC inhibitors on the reactivation of latent HIV-1 were determined and compared with anti-CD3 and anti-CD28 co-stimulation. RESULTS: In Bcl-2-transduced primary CD4+ T cells, short-term treatment with HDAC inhibitors resulted in very limited reactivation of latent HIV-1, while prolonged treatment greatly enhanced drug efficacy. The effects of HDAC inhibitors in reactivating latent HIV-1 correlated with their inhibitory effects on class I HDACs. Importantly, HIV-1 reactivated by HDAC inhibitors can quickly re-establish latent infection upon drug removal. CONCLUSIONS: We identified unique features of HDAC inhibitor-induced reactivation of latent HIV-1 in primary CD4+ T cells. Our findings may be useful for the design of eradication trials.

Targeting HIV latency: pharmacologic strategies toward eradication.

The latent reservoir for HIV-1 in resting CD4(+) T cells remains a major barrier to HIV-1 eradication, even though highly active antiretroviral therapy (HAART) can successfully reduce plasma HIV-1 levels to below the detection limit of clinical assays and reverse disease progression. Proposed eradication strategies involve reactivation of this latent reservoir. Multiple mechanisms are believed to be involved in maintaining HIV-1 latency, mostly through suppression of transcription. These include cytoplasmic sequestration of host transcription factors and epigenetic modifications such as histone deacetylation, histone methylation and DNA methylation. Therefore, strategies targeting these mechanisms have been explored for reactivation of the latent reservoir. In this review, we discuss current pharmacological approaches toward eradication, focusing on small molecule latency-reversing agents, their mechanisms, advantages and limitations.

BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism.

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.

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.

Novel structurally related compounds reactivate latent HIV-1 in a bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation.

BACKGROUND: The latent reservoir of HIV-1 in resting memory CD4+ T cells is a major barrier to curing HIV-1 infection. Eradication strategies involve reactivation of this latent reservoir; however, agents that reactivate latent HIV-1 through non-specific T cell activation are toxic. METHODS: Using latently infected Bcl-2-transduced primary CD4+ T cells, we screened the MicroSource Spectrum library for compounds that reactivate latent HIV-1 without global T cell activation. Based on the structures of the initial hits, we assembled ∼50 derivatives from commercial sources and mostly by synthesis. The dose-response relationships of these derivatives were established in a primary cell model. Activities were confirmed with another model of latency (J-Lat). Cellular toxicity and cytokine secretion were tested using freshly isolated human CD4+ T cells. RESULTS: We identified two classes of quinolines that reactivate latent HIV-1. Class I compounds are the Mannich adducts of 5-chloroquinolin-8-ol. Class II compounds are quinolin-8-yl carbamates. Most EC(50) values were in the 0.5-10 µM range. HIV-1 reactivation ranged from 25% to 70% for anti-CD3+ anti-CD28 co-stimulation. All quinolin-8-ol derivatives that reactivate latent HIV-1 follow Lipinski's Rule of Five, and most follow the stricter rule of three for leads. After 48 h of treatment, none of the analogues induced detectable cytokine secretion in primary resting CD4+ T cells. CONCLUSIONS: We discovered a group of quinolin-8-ol derivatives that can induce latent HIV-1 in a primary cell model without causing global T cell activation. This work expands the number of latency-reversing agents and provides new possible scaffolds for further drug development research.

A critical subset model provides a conceptual basis for the high antiviral activity of major HIV drugs.

Control of HIV-1 replication was first achieved with regimens that included a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI); however, an explanation for the high antiviral activity of these drugs has been lacking. Indeed, conventional pharmacodynamic measures like IC(50) (drug concentration causing 50% inhibition) do not differentiate NNRTIs and PIs from less active nucleoside reverse transcriptase inhibitors (NRTIs). Drug inhibitory potential depends on the slope of the dose-response curve (m), which represents how inhibition increases as a function of increasing drug concentration and is related to the Hill coefficient, a measure of intramolecular cooperativity in ligand binding to a multivalent receptor. Although NNRTIs and PIs bind univalent targets, they unexpectedly exhibit cooperative dose-response curves (m > 1). We show that this cooperative inhibition can be explained by a model in which infectivity requires participation of multiple copies of a drug target in an individual life cycle stage. A critical subset of these target molecules must be in the unbound state. Consistent with experimental observations, this model predicts m > 1 for NNRTIs and PIs and m = 1 in situations where a single drug target/virus mediates a step in the life cycle, as is the case with NRTIs and integrase strand transfer inhibitors. This model was tested experimentally by modulating the number of functional drug targets per virus, and dose-response curves for modulated virus populations fit model predictions. This model explains the high antiviral activity of two drug classes important for successful HIV-1 treatment and defines a characteristic of good targets for antiviral drugs in general, namely, intermolecular cooperativity.

Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation.

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4(+) cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4(+) T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.

Small-molecule screening using a human primary cell model of HIV latency identifies compounds that reverse latency without cellular activation.

The development of highly active antiretroviral therapy (HAART) to treat individuals infected with HIV-1 has dramatically improved patient outcomes, but HAART still fails to cure the infection. The latent viral reservoir in resting CD4+ T cells is a major barrier to virus eradication. Elimination of this reservoir requires reactivation of the latent virus. However, strategies for reactivating HIV-1 through nonspecific T cell activation have clinically unacceptable toxicities. We describe here the development of what we believe to be a novel in vitro model of HIV-1 latency that we used to search for compounds that can reverse latency. Human primary CD4+ T cells were transduced with the prosurvival molecule Bcl-2, and the resulting cells were shown to recapitulate the quiescent state of resting CD4+ T cells in vivo. Using this model system, we screened small-molecule libraries and identified a compound that reactivated latent HIV-1 without inducing global T cell activation, 5-hydroxynaphthalene-1,4-dione (5HN). Unlike previously described latency-reversing agents, 5HN activated latent HIV-1 through ROS and NF-kappaB without affecting nuclear factor of activated T cells (NFAT) and PKC, demonstrating that TCR pathways can be dissected and utilized to purge latent virus. Our study expands the number of classes of latency-reversing therapeutics and demonstrates the utility of this in vitro model for finding strategies to eradicate HIV-1 infection.

The HBV drug entecavir - effects on HIV-1 replication and resistance.

Entecavir, a drug approved by the Food and Drug Administration for the treatment of chronic hepatitis B virus (HBV) infection, is not believed to inhibit replication of human immunodeficiency virus type 1 (HIV-1) at clinically relevant doses. We observed that entecavir led to a consistent 1-log(10) decrease in HIV-1 RNA in three persons with HIV-1 and HBV coinfection, and we obtained supportive in vitro evidence that entecavir is a potent partial inhibitor of HIV-1 replication. Detailed analysis showed that in one of these patients, entecavir monotherapy led to an accumulation of HIV-1 variants with the lamivudine-resistant mutation, M184V. In vitro experiments showed that M184V confers resistance to entecavir. Until more is known about HIV-1-resistance patterns and their selection by entecavir, caution is needed with the use of entecavir in persons with HIV-1 and HBV coinfection who are not receiving fully suppressive antiretroviral regimens.