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.

Background The recent discovery of the role of m6A methylation in the regulation of HIV-1 replication unveiled a novel layer of regulation for HIV gene expression. This epitranscriptomic modification of HIV-1 RNAs is under the dynamic control of specific writers and erasers. In addition, cytoplasmic readers of the m6A mark are recruited to the modified viral RNAs and regulate HIV-1 replication. Yet, little is known about the effects of m6A writers and readers on the biogenesis of HIV-1 RNAs. Results We showed that the METTL3/14 m6A methyltransferase complex and the m6A YTHDF2 cytoplasmic writer down regulates the abundance of HIV-1 RNAs in infected cells. We also identified the m6A nuclear writer YTHDC1 as a novel regulator of HIV-1 transcripts. In HIV-1 producer cells, we showed that knocking down YTHDC1 increases the levels of unspliced and incompletely spliced HIV-1 RNAs, while levels of multiply spliced transcripts remained unaffected. In addition, we observed that depletion of YTHDC1 has no effect on the nuclear cytoplasmic distribution of viral transcripts. YTHDC1 binds specifically to HIV-1 transcripts in a METTL3-dependent manner. Knocking down YTHDC1 reduces the expression of Env and Vpu viral proteins in producer cells and leads to the incorporation of unprocessed Env gp160 in virus particles, resulting in the decrease of their infectivity. Conclusions Our findings indicate that, by controlling HIV-1 RNA biogenesis and protein expression, the m6A nuclear reader YTHDC1 is required for efficient production of infectious viral particles. Graphical Abstract

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The multiplicity, heterogeneity and dynamic nature of HIV-1 latency mechanisms are reflected in the current lack of functional cure for HIV-1 and in the various reported ex vivo potencies of latency-reversing agents. Here, we investigated the molecular mechanisms underlying the potency of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC) in HIV-1 latency reversal. Doing so, 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 UHRF1 to the HIV-1 promoter. We further demonstrated the role of UHRF1 in DNA methylation-mediated silencing of the latent HIV-1 promoter. As a proof-of-concept to this molecular characterization, we showed that pharmacological downregulation of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent reactivation of latent HIV-1. Together, we identify UHRF1 as a novel actor in HIV-1 gene silencing and highlight that it constitutes a new molecular target for HIV-1 curative strategies.