The BET bromodomain inhibitor apabetalone induces apoptosis of latent HIV-1 reservoir cells following viral reactivation

ABSTRACT Antiretroviral therapy (ART) suppresses HIV-1 replication but fails to cure the infection. The presence of an extremely stable viral latent reservoir, primarily in resting memory CD4+ T cells, remains a major obstacle to viral eradication. The “shock and kill” strategy targets these latently infected cells and boosts immune recognition and clearance, and thus, it is a promising approach for an HIV-1 functional cure. Although some latency-reversing agents (LRAs) have been reported, no apparent clinical progress has been made, so it is still vital to seek novel and effective LRAs. Here, we report that thiostrepton (TSR), a proteasome inhibitor, reactivates latent HIV-1 effectively in cellular models and in primary CD4+ T cells from ART-suppressed individuals ex vivo. TSR does not induce global T cell activation, severe cytotoxicity, or CD8+ T cell dysfunction, making it a prospective LRA candidate. We also observed a significant synergistic effect of reactivation when TSR was combined with JQ1, prostratin, or bryostatin-1. Interestingly, six TSR analogues also show reactivation abilities that are similar to or more effective than that of TSR. We further verified that TSR upregulated expression of heat shock proteins (HSPs) in CD4+ T cells, which subsequently activated positive transcriptional elongation factor b (p-TEFb) and NF-κB signals, leading to viral reactivation. In summary, we identify TSR as a novel LRA which could have important significance for applications to an HIV-1 functional cure in the future.

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A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other “Shock” Drugs

Journal of Virology ◽

10.1128/jvi.02056-17 ◽

2018 ◽

Vol 92 (10) ◽

Cited By ~ 10

Author(s):

Erik Abner ◽

Mateusz Stoszko ◽

Lei Zeng ◽

Heng-Chang Chen ◽

Andrea Izquierdo-Bouldstridge ◽

...

Keyword(s):

Hdac Inhibitors ◽

Jurkat Cells ◽

Viral Reactivation ◽

Viral Reservoir ◽

Latently Infected ◽

Core Histones ◽

Latent Hiv ◽

Expression Microarrays ◽

Hiv 1

ABSTRACT Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or “shock” drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection. IMPORTANCE The suggested “shock and kill” therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the “shocking” element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo . Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

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Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

10.1101/2020.12.30.424810 ◽

2020 ◽

Author(s):

Iart Luca Shytaj ◽

Francesco Andrea Procopio ◽

Mohammad Tarek ◽

Irene Carlon-Andres ◽

Hsin-Yao Tang ◽

...

Keyword(s):

Pentose Phosphate ◽

Viral Reactivation ◽

People Living With Hiv ◽

Cellular Models ◽

Downstream Effectors ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Living With Hiv ◽

Hiv 1

AbstractHIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

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CARD8 is an inflammasome sensor for HIV-1 protease activity

Science ◽

10.1126/science.abe1707 ◽

2021 ◽

Vol 371 (6535) ◽

pp. eabe1707 ◽

Cited By ~ 2

Author(s):

Qiankun Wang ◽

Hongbo Gao ◽

Kolin M. Clark ◽

Christian Shema Mugisha ◽

Keanu Davis ◽

...

Keyword(s):

Protease Activity ◽

Rapid Evolution ◽

Viral Persistence ◽

Viral Reactivation ◽

Mutation Rates ◽

Host Immune System ◽

Viral Budding ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain–containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome–mediated pyroptosis of HIV-1–infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for the clearance of persistent HIV-1 infection.

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Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

10.1101/2020.08.25.265413 ◽

2020 ◽

Cited By ~ 1

Author(s):

Katiana Tantale ◽

Encarnation Garcia-Oliver ◽

Adèle L’Hostis ◽

Yueyuxio Yang ◽

Marie-Cécile Robert ◽

...

Keyword(s):

Single Molecule ◽

Viral Reactivation ◽

Multiple Time Scales ◽

Viral Transcription ◽

Multiple Time ◽

Quantitative Analysis Method ◽

Polymerase Pausing ◽

Latent Hiv ◽

Transcriptional Bursting ◽

Hiv 1

SummaryPromoter-proximal polymerase pausing is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription, and we developed a quantitative analysis method that manages multiple time scales from seconds to days, and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.

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HIV Type-1 Latency: Targeted Induction of Proviral Reservoirs

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020901900501 ◽

2009 ◽

Vol 19 (5) ◽

pp. 177-187 ◽

Cited By ~ 5

Author(s):

Jason D Graci ◽

Joseph M Colacino ◽

Stuart W Peltz ◽

Joseph P Dougherty ◽

Zhengxian Gu

Keyword(s):

T Cells ◽

Highly Active Antiretroviral Therapy ◽

Current Knowledge ◽

Therapeutic Agents ◽

Viral Reactivation ◽

Highly Active ◽

Hiv Type 1 ◽

Latent Hiv ◽

Hiv 1

HIV type-1 (HIV-1) can establish a state of latency in infected patients, most notably in resting CD4+ T-cells. This long-lived reservoir allows for rapid re-emergence of viraemia upon cessation of highly active antiretroviral therapy, even after extensive and seemingly effective treatment. Successful depletion of such latent reservoirs is probably essential to ‘cure’ HIV-1 infection and will require therapeutic agents that can specifically and efficiently act on cells harbouring latent HIV-1 provirus. The mechanisms underlying HIV-1 latency are not well characterized, and it is becoming clear that numerous factors, both cell- and virus-derived, are involved in the maintenance of proviral latency. The interplay of these various factors in the context of viral reactivation is still poorly understood. In this article, we review the current knowledge regarding the mechanisms underlying maintenance of HIV-1 latency, both transcriptional and post-transcriptional, with a focus on potential targets that might be exploited to therapeutically purge latent proviral reservoirs from infected patients.

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Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

Nature Communications ◽

10.1038/s41467-021-24462-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katjana Tantale ◽

Encar Garcia-Oliver ◽

Marie-Cécile Robert ◽

Adèle L’Hostis ◽

Yueyuxiao Yang ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Single Molecule ◽

Viral Reactivation ◽

Multiple Time Scales ◽

Viral Transcription ◽

Multiple Time ◽

Quantitative Analysis Method ◽

Latent Hiv ◽

Transcriptional Bursting ◽

Hiv 1

AbstractPromoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.

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Maraviroc Is Associated with Latent HIV-1 Reactivation through NF-κB Activation in Resting CD4+T Cells from HIV-Infected Individuals on Suppressive Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.01931-17 ◽

2018 ◽

Vol 92 (9) ◽

Cited By ~ 23

Author(s):

Nadia Madrid-Elena ◽

María Laura García-Bermejo ◽

Sergio Serrano-Villar ◽

Alberto Díaz-de Santiago ◽

Beatriz Sastre ◽

...

Keyword(s):

T Cells ◽

Antiretroviral Therapy ◽

New Drugs ◽

Viral Reactivation ◽

Ccr5 Antagonist ◽

Reversal Agent ◽

Open Label ◽

Major Barrier ◽

Latent Hiv ◽

Hiv 1

ABSTRACTMaraviroc is a CCR5 antagonist used in the treatment of HIV-1 infection. We and others have suggested that maraviroc could reactivate latent HIV-1. To test the latency-reversing potential of maraviroc and the mechanisms involved, we performed a phase II, single-center, open-label study in which maraviroc was administered for 10 days to 20 HIV-1-infected individuals on suppressive antiretroviral therapy (EudraCT registration no. 2012-003215-66). All patients completed full maraviroc dosing and follow-up. The primary endpoint was to study whether maraviroc may reactivate HIV-1 latency, eliciting signaling pathways involved in the viral reactivation. An increase in HIV-1 transcription in resting CD4+T cells, estimated by levels of HIV-1 unspliced RNA, was observed. Moreover, activation of the NF-κB transcription factor was observed in these cells. To elucidate the mechanism of NF-κB activation by maraviroc, we have evaluated in HeLa P4 C5 cells, which stably express CCR5, whether maraviroc could be acting as a partial CCR5 agonist, with no other mechanisms or pathways involved. Our results show that maraviroc can induce NF-κB activity and that NF-κB targets gene expression by CCR5 binding, since the use of TAK779, a CCR5 inhibitor, blocked NF-κB activation and functionality. Taking the results together, we show that maraviroc may have a role in the activation of latent virus transcription through the activation of NF-κB as a result of binding CCR5. Our results strongly support a novel use of maraviroc as a potential latency reversal agent in HIV-1-infected patients.IMPORTANCEHIV-1 persistence in a small pool of long-lived latently infected resting CD4+T cells is a major barrier to viral eradication in HIV-1-infected patients on antiretroviral therapy. A potential strategy to cure HIV-1-infection is the use of latency-reversing agents to eliminate the reservoirs established in resting CD4+T cells. As no drug has been shown to be completely effective so far, the search for new drugs and combinations remains a priority for HIV cure. We examined the ability of maraviroc, a CCR5 antagonist used as an antiretroviral drug, to activate latent HIV-1 in infected individuals on antiretroviral therapy. The study showed that maraviroc can activate NF-κB and, subsequently, induce latent HIV-1-transcription in resting CD4+T cells from HIV-1-infected individuals on suppressive antiretroviral therapy. Additional interventions will be needed to eliminate latent HIV-1 infection. Our results suggest that maraviroc may be a new latency-reversing agent to interfere with HIV-1 persistence during antiretroviral therapy.

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