Nadège Delacourt

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.

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.

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 serine473 and threonine308. 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.

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.

In vivo characterization of the BLV miRNA cluster

Bovine leukemia virus (BLV), a B-lymphotropic oncogenic retrovirus sharing common biological and structural features with the human T-cell leukemia virus I and II (HTLV-I and II), is the etiologic agent of enzootic bovine leucosis. One major feature of the BLV infection is the absence of viremia. It is widely accepted that BLV latency, due to the RN! polymerase II 5’-LTR-driven transcriptional and epigenetic repression is a viral strategy to escape from the host immune system and allow tumor development. Recently, it has been demonstrated by deep sequencing and bioinformatics analysis that the BLV genome encodes a cluster of micro-RNAs which is predicted to be transcribed by RNA polymerase III suggesting that the silencing dogma in BLV transcriptional regulation is only partially correct. Here, we demonstrated by chromatin immunoprecipitation assays that BLV miRNAs were indeed transcribed in vivo by RNA polymerase III through a type II RNAPIII promoter similar to the one directing tRNAs transcription. We also showed that an activating epigenetic environment surrounds the miRNAs cluster. Overall, our results provide new insights into a better understanding of the molecular mechanisms regulating gene expression of oncogenic retroviruses.

ARE-mediated decay controls gene expression and cellular metabolism upon oxygen variations

Hypoxia triggers profound modifications of cellular transcriptional programs. Upon reoxygenation, cells return to a normoxic gene expression pattern and mRNA produced during the hypoxic phase are degraded. TIS11 proteins control deadenylation and decay of transcripts containing AU-rich elements (AREs). We observed that the level of dTIS11 is decreased in hypoxic S2 Drosophila cells and returns to normal level upon reoxygenation. Bioinformatic analyses using the ARE-assessing algorithm AREScore show that the hypoxic S2 transcriptome is enriched in ARE-containing transcripts and that this trend is conserved in human myeloid cells. Moreover, an efficient down-regulation of Drosophila ARE-containing transcripts during hypoxia/normoxia transition requires dtis11 expression. Several of these genes encode proteins with metabolic functions. Here, we show that ImpL3 coding for Lactate Dehydrogenase in Drosophila, is regulated by ARE-mediated decay (AMD) with dTIS11 contributing to ImpL3 rapid down-regulation upon return to normal oxygen levels after hypoxia. More generally, we observed that dtis11 expression contributes to cell metabolic and proliferative recovery upon reoxygenation. Altogether, our data demonstrate that AMD plays an important role in the control of gene expression upon variation in oxygen concentration and contributes to optimal metabolic adaptation to oxygen variations.

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.