Reactivation of latent HIV-1 by new semi-synthetic ingenol esters.
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ABSTRACT:
SUBMITTER: Pandelo Jose D
PROVIDER: S-EPMC4383768 | biostudies-literature | 2014 Aug
REPOSITORIES: biostudies-literature
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Project description:HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-?B activators Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection.
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Project description:Long-lived pools of latently infected cells are a significant barrier to the development of a cure for HIV-1 infection. A better understanding of the mechanisms of reactivation from latency is needed to facilitate the development of novel therapies that address this problem. Here we show that chemical inhibitors of the sulfonation pathway prevent virus reactivation, both in latently infected J-Lat and U1 cell lines and in a primary human CD4+ T cell model of latency. In each of these models, sulfonation inhibitors decreased transcription initiation from the HIV-1 promoter. These inhibitors block transcription initiation at a step that lies downstream of nucleosome remodeling and affects RNA polymerase II recruitment to the viral promoter. These results suggest that the sulfonation pathway acts by a novel mechanism to regulate efficient virus transcription initiation during reactivation from latency, and further that augmentation of this pathway could be therapeutically useful.
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Project description:Actinic keratosis (AK) is a precancerous skin lesion that is common in HIV-positive patients. Without effective treatment, AKs can progress to squamous cell carcinoma. Ingenol mebutate, a PKC agonist, is a US Food and Drug Administration-approved (FDA-approved) topical treatment for AKs. It can induce reactivation of latent HIV transcription in CD4+ T cells both in vitro and ex vivo. Although PKC agonists are known to be potent inducers of HIV expression from latency, their effects in vivo are not known because of the concerns of toxicity. Therefore, we sought to determine the effects of topical ingenol mebutate gel on the HIV transcription profile in HIV-infected individuals with AKs, specifically in the setting of suppressive antiretroviral therapy (ART). We found that AKs cleared following topical application of ingenol mebutate and detected marginal changes in immune activation in the peripheral blood and in skin biopsies. An overall increase in the level of HIV transcription initiation, elongation, and complete transcription was detected only in skin biopsies after the treatment. Our data demonstrate that application of ingenol mebutate to AKs in ART-suppressed HIV-positive patients can effectively cure AKs as well as disrupt HIV latency in the skin tissue microenvironment in vivo without causing massive immune activation.
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Project description:The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.
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Project description:HIV-1 escapes antiretroviral drugs by integrating into the host DNA and forming a latent transcriptionally silent HIV-1 provirus. This provirus presents the major hurdle in HIV-1 eradication and cure. Transcriptional activation, which is prerequisite for reactivation and the eradication of latent proviruses, is impaired in latently infected T cells due to the lack of host transcription factors, primarily NF-?B and P-TEFb (CDK9/cyclin T1). We and others previously showed that protein phosphatase-1 (PP1) regulates HIV-1 transcription by modulating CDK9 phosphorylation. Recently we have developed a panel of small molecular compounds targeting a non-catalytic site of PP1.Here we generated a new class of sulfonamide-containing compounds that activated HIV-1 in acute and latently infected cells. Among the tested molecules, a small molecule activator of PP1 (SMAPP1) induced both HIV-1 replication and reactivation of latent HIV-1 in chronically infected cultured and primary cells. In vitro, SMAPP1 interacted with PP1 and increased PP1 activity toward a recombinant substrate. Treatment with SMAPP1 increased phosphorylation of CDK9's Ser90 and Thr186 residues, but not Ser175. Proteomic analysis showed upregulation of P-TEFb and PP1 related proteins, including PP1 regulatory subunit Sds22 in SMAPP1-treated T cells. Docking analysis identified a PP1 binding site for SMAPP1 located within the C-terminal binding pocket of PP1.We identified a novel class of PP1-targeting compounds that reactivate latent HIV-1 provirus by targeting PP1, increasing CDK9 phosphorylation and enhancing HIV transcription. This compound represents a novel candidate for anti-HIV-1 therapeutics aiming at eradication of latent HIV-1 reservoirs.
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Project description:Cell state switches underlie a plethora of biological phenomena and disease treatment strategies. Hence the ability to efficiently switch states in a chosen direction is of central importance in a number of scenarios. Increasing the concentration of an effector that results in a given switch is often limited by side effects. Approaches are thus increasingly sought to bypass these constraints, increasing the frequency of state switching without increasing the frequency of the side effect. Here, we employ dynamical systems theory to uncover a simple strategy as to how to maximize the probability of reactivating latent Human immunodeficiency virus (HIV) whilst maintaining minimal side effects. We demonstrate that continuous supply of an effector is significantly more likely to result in a switch with minimal side effects than the same effector supplied in temporally discrete doses. Importantly this continual dosage is likely to occur far below the Minimum effective dose at a concentration that has classically been thought subtherapeutic. We therefore suggest that in many interventional settings there exists potential to reduce drug dose much further than has previously been thought possible yet still maintaining efficacy.
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Project description:The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.
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Project description: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.
| S-EPMC5923069 | biostudies-literature