Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.

BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.

[Viral latency of HIV-1]. / La latence virale du VIH-1.

Human immunodeficiency virus (HIV-1) latency is clinically highlighting via the persistence of a residual viral load in cART-treated patients due to the reactivation of cellular reservoirs. Two forms of latency coexist but the contribution of the pre-integrationnal latency clearly plays a minor role in viral persistence. In contrast, the post-integrationnal latency significantly contributes to the evasion of the immune system by the HIV-1 cellular reservoir and consequently to HIV-1 pathogenesis. Although post-transcriptional mechanisms can contribute to the maintenance of viral latency, HIV-1 transcriptional inhibition is critical for the establishment and maintenance of post-integrational latency. This inhibition is a multifactorial phenomenon, making the development of anti-latency therapeutic strategies complex. These different notions will be described throughout this review.

Heterogeneous HIV-1 Reactivation Patterns of Disulfiram and Combined Disulfiram+Romidepsin Treatments.

OBJECTIVES: Few single latency-reversing agents (LRAs) have been tested in vivo, and only some of them have demonstrated an effect, albeit weak, on the decrease of latent reservoir. Therefore, other LRAs and combinations of LRAs need to be assessed. Here, we evaluated the potential of combined treatments of therapeutically promising LRAs, disulfiram and romidepsin. SETTING AND METHODS: We assessed the reactivation potential of individual disulfiram or simultaneous or sequential combined treatments with romidepsin in vitro in latently infected cell lines of T-lymphoid and myeloid origins and in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells isolated from 18 HIV-1 combination antiretroviral therapy-treated individuals. RESULTS: We demonstrated heterogeneous reactivation effects of disulfiram in vitro in various cell lines of myeloid origin and no latency reversal neither in vitro in T-lymphoid cells nor ex vivo, even if doses corresponding to maximal plasmatic concentration or higher were tested. Disulfiram+romidepsin combined treatments produced distinct reactivation patterns in vitro. Ex vivo, the combined treatments showed a modest reactivation effect when used simultaneously as opposed to no viral reactivation for the corresponding sequential treatment. CONCLUSIONS: Exclusive reactivation effects of disulfiram in myeloid latency cell lines suggest that disulfiram could be a potential LRA for this neglected reservoir. Moreover, distinct reactivation profiles pinpoint heterogeneity of the latent reservoir and confirm that the mechanisms that contribute to HIV latency are diverse. Importantly, disulfiram+romidepsin treatments are not potent ex vivo and most likely do not represent an effective drug combination to achieve high levels of latency reversal in vivo.

The Molecular Biology of HIV Latency.

HIV remains incurable due to the existence of a reservoir of cells that harbor intact integrated genomes of the virus in the absence of viral replication. This population of infected cells remains invisible to the immune system and is not targeted by the drugs used in the current antiretroviral therapies (cART). Reversal of latency by the use of inhibitors of chromatin-remodeling enzymes has been studied extensively in an attempt to purge this reservoir of latent HIV but has thus far not shown any success in clinical trials. The full complexity of latent HIV infection has still not been appreciated, and the gaps in knowledge prevent development of adequate small-molecule compounds that can effectively perturb this reservoir. In this review, we will examine the role of epigenetic silencing of HIV transcription, posttranscriptional regulation, and mRNA processing in promoting HIV-1 latency.

Immunity drives TET1 regulation in cancer through NF-κB.

Ten-eleven translocation enzymes (TET1, TET2, and TET3), which induce DNA demethylation and gene regulation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), are often down-regulated in cancer. We uncover, in basal-like breast cancer (BLBC), genome-wide 5hmC changes related to TET1 regulation. We further demonstrate that TET1 repression is associated with high expression of immune markers and high infiltration by immune cells. We identify in BLBC tissues an anticorrelation between TET1 expression and the major immunoregulator family nuclear factor κB (NF-κB). In vitro and in mice, TET1 is down-regulated in breast cancer cells upon NF-κB activation through binding of p65 to its consensus sequence in the TET1 promoter. We lastly show that these findings extend to other cancer types, including melanoma, lung, and thyroid cancers. Together, our data suggest a novel mode of regulation for TET1 in cancer and highlight a new paradigm in which the immune system can influence cancer cell epigenetics.

Molecular Control of HIV and SIV Latency.

The HIV latent reservoirs are considered as the main hurdle to viral eradication. Numerous mechanisms lead to the establishment of HIV latency and act at the transcriptional and post-transcriptional levels. A better understanding of latency is needed in order to ultimately achieve a cure for HIV. The mechanisms underlying latency vary between patients, tissues, anatomical compartments, and cell types. From this point of view, simian immunodeficiency virus (SIV) infection and the use of nonhuman primate (NHP) models that recapitulate many aspects of HIV-associated latency establishment and disease progression are essential tools since they allow extensive tissue sampling as well as a control of infection parameters (virus type, dose, route, and time).

On the way to find a cure: Purging latent HIV-1 reservoirs.

Introduction of cART in 1996 has drastically increased the life expectancy of people living with HIV-1. However, this treatment has not allowed cure as cessation of cART is associated with a rapid viral rebound. The main barrier to the eradication of the virus is related to the persistence of latent HIV reservoirs. Evidence is now accumulating that purging the HIV-1 reservoir might lead to a cure or a remission. The most studied strategy is the so called "shock and kill" therapy. This strategy is based on reactivation of dormant viruses from the latently-infected reservoirs (the shock) followed by the eradication of the reservoirs (the kill). This review focuses mainly on the recent advances made in the "shock and kill" therapy. We believe that a cure or a remission will come from combinatorial approaches i.e. combination of drugs to reactivate the dormant virus from all the reservoirs including the one located in sanctuaries, and combination of strategies boosting the immune system. Alternative strategies based on cell and gene therapy or based in inducing deep latency, which are evoked in this review reinforce the idea that at least a remission is attainable.

Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites.

Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.

Reactivation capacity by latency-reversing agents ex vivo correlates with the size of the HIV-1 reservoir.

OBJECTIVE: HIV-1 reservoirs are the major hurdle to virus clearance in combination antiretroviral therapy (cART)-treated patients. An approach to eradicating HIV-1 involves reversing latency in cART-treated patients to make latent cells visible to the host immune system. Stimulation of patient cell cultures with latency-reversing agents (LRAs) ex vivo results in heterogeneous responses among HIV-infected patients. Determinants of this heterogeneity are unknown and consequently important to determine. DESIGN AND METHODS: Here, we grouped and retrospectively analyzed the data from our two recent HIV-1 reactivation studies to investigate the role of the HIV-1 reservoir size in the reactivation capacity by LRAs in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells (PBMCs) isolated from 54 cART-treated patients and of resting CD4 T cells isolated from 30 cART-treated patients. RESULTS: Our results established a statistically relevant positive correlation between the HIV-1 reservoir size measured by total cell-associated HIV-1 DNA and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of cells isolated from cART-treated HIV aviremic patients. HIV-1 reservoir size also correlated with the extracellular HIV-1 RNA median level measured in supernatants of cell cultures following LRA treatments. However, we identified HIV patients whose positive measurements frequency and median level of extracellular HIV-1 RNA deviated from linearity relative to their corresponding HIV reservoir size. CONCLUSION: We demonstrated that the reservoir size is one predictive marker of LRA effectiveness but this parameter alone is not sufficient. The identification of other predictive markers is necessary to predict the success of HIV anti-latency approaches.

Limited HIV-1 Reactivation in Resting CD4+ T cells from Aviremic Patients under Protease Inhibitors.

A latent viral reservoir that resides in resting CD4+ T cells represents a major barrier for eradication of HIV infection. We test here the impact of HIV protease inhibitor (PI) based combination anti-retroviral therapy (cART) over nonnucleoside reverse transcriptase inhibitor (NNRTI)-based cART on HIV-1 reactivation and integration in resting CD4+ T cells. This is a prospective cohort study of patients with chronic HIV-1 infection treated with conventional cART with an undetectable viremia. We performed a seven-year study of 47 patients with chronic HIV-infection treated with cART regimens and with undetectable plasma HIV-1 RNA levels for at least 1 year. Of these 47 patients treated with cART, 24 were treated with a PI-based regimen and 23 with a NNRTI-based regimen as their most recent treatment for more than one year. We evaluated the HIV-1 reservoir using reactivation assay and integrated HIV-1 DNA, respectively, in resting CD4+ T cells. Resting CD4+ T cells isolated from PI-treated patients compared to NNRTI-treated patients showed a limited HIV-1 reactivation upon T-cell stimulation (p = 0·024) and a lower level of HIV-1 integration (p = 0·024). Our study indicates that PI-based cART could be more efficient than NNRTI-based cART for limiting HIV-1 reactivation in aviremic chronically infected patients.

Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.

Sequential treatment with 5-aza-2'-deoxycytidine and deacetylase inhibitors reactivates HIV-1.

Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency-reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5-AzadC) in combination with clinically tolerable HDACIs in reactivating HIV-1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5-AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV-1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5-AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

HIV-1 transcription and latency: an update.

Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs.

Histone methyltransferase inhibitors induce HIV-1 recovery in resting CD4(+) T cells from HIV-1-infected HAART-treated patients.

OBJECTIVE: Reactivation of HIV-1 expression in persistent reservoirs together with an efficient HAART has been proposed as an adjuvant therapy aimed at reaching a functional cure for HIV. Previously, H3K9 methylation was shown to play a major role in chromatin-mediated repression of the HIV-1 promoter. Here, we evaluated the therapeutic potential of histone methyltransferase inhibitors (HMTIs) in reactivating HIV-1 from latency. DESIGN: We evaluated the reactivation potential of two specific HMTIs (chaetocin and BIX-01294, two specific inhibitors of Suv39H1 and G9a, respectively) in ex-vivo cultures of resting CD4 T cells isolated from HIV-1-infected HAART-treated individuals. METHODS: We measured HIV-1 recovery in ex-vivo cultures treated with an HMTI alone or in combination with other HIV-1 inducers (in absence of IL-2 and of allogenic stimulation) of CD8-depleted peripheral blood mononuclear cells (PBMCs) or of resting CD4 T cells isolated from 67 HIV-infected, HAART-treated patients with undetectable viral load. RESULTS: We demonstrated, for the first time, that chaetocin induced HIV-1 recovery in 50% of CD8-depleted PBMCs cultures and in 86% of resting CD4 T-cell cultures isolated from HIV-1-infected, HAART-treated patients, whereas BIX-01294 reactivated HIV-1 expression in 80% of resting CD4 T-cell cultures isolated from similar patients. Moreover, we showed that combinatory treatments including one HMTI and either the histone deacetylase inhibitor suberoylanilide hydroxamic acid or the non-tumor-promoting NF-κB inducer prostratin had a higher reactivation potential than these compounds alone. CONCLUSION: Our results constitute a proof-of-concept for the therapeutic potential of HMTIs in strategies aiming at reducing the pool of latent reservoirs in HIV-infected, HAART-treated patient.