Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (M^pro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent M^pro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC₅₀ of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for M^pro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher M^pro activity of 2d and, as a result, its better antiviral profile.

The "shock-and-kill" strategy is one of the most explored HIV-1 cure approaches to eliminate latent virus. This strategy is based on HIV-1 reactivation using latency reversing agents (LRAs) to reactivate latent proviruses (the "shock" phase) and to induce subsequent elimination of the reactivated cells by immune responses or virus-induced cytopathic effects (the "kill" phase). Studies using immunomodulatory LRAs such as blockers of immune checkpoint molecules, toll-like receptor agonists, cytokines and CD8+ T cell depleting antibodies showed promising potential as LRAs inducing directly or indirectly cellular pathways known to control HIV transcription. However, the precise molecular mechanisms by which these immunomodulatory LRAs reverse latency remain incompletely understood. Together with the heterogenous nature of HIV-1 latency, this lack of understanding complicates efforts to develop more efficient and safer cure strategies. Hence, deciphering those mechanisms is pivotal in designing approaches to eliminate latent HIV infection.

HIV-1 latency is mainly characterized at transcriptional level, and little is known about post-transcriptional mechanisms and their contribution to reactivation. The viral protein Rev controls the nucleocytoplasmic export of unspliced and singly-spliced RNA that is central to proviral replication-competence and is therefore a prerequisite for efficient viral reactivation during the “shock-and-kill” cure therapy. Here we show that during infection and reactivation, unspliced HIV-1 RNA is a subject to complex and dynamic regulation by the Rev cofactor MATR3 and the MTR4 cofactor of the nuclear exosome. MATR3 and MTR4 coexist in the same ribonucleoprotein complex functioning to either maintain or degrade the RNA, respectively, with Rev orchestrating this regulatory switch. Moreover, we provide evidence of nuclear retention of unspliced HIV-1 RNA in ex vivo cultures from 22 ART-treated people with HIV, highlighting a reversible post-transcriptional block to viral RNA nucleocytoplasmic export that is relevant to the design of curative interventions.

Current latency-reversing agents (LRAs) employed in the “shock-and-kill” strategy primarily focus on relieving epigenetic and transcriptional blocks to reactivate the latent HIV-1. However, their clinical efficacy is limited, partly due to their inability to fully reverse latency and the lack of LRAs specifically targeting post-transcriptional mechanisms. N6-methyladenosine (m6A) modification in HIV-1 RNA is emerging as an important post-transcriptional regulator of HIV-1 gene expression, yet its role in latency and reactivation remains largely unrecognized. Here, we explored the potential of small chemical compounds targeting the m6A pathway, specifically investigating the inhibition of ALKBH5 and its effect on latent HIV-1 reactivation mediated by the LRA romidepsin. We used four in vitro cellular models of latency, primary model of CD4+ T cells HIV-1 infection and ex vivo cultures of CD8+-depleted PMBCs from ART-treated HIV+ patients. We measured latent viral reactivation by evaluating the expression of reporter protein GFP by flow cytometry, viral production by CA-p24 ELISA, and viral transcripts by RT-qPCR. CRISPR/Cas9 method was used to deplete ALKBH5. MeRIP and immuno-RNA FISH were used to address the m6A methylation levels on HIV-1 RNA upon ALKBH5 inhibition. We showed that ALKBH5 inhibitor 3 (ALKi-3) potentiated romidepsin-mediated viral reactivation in in vitro models of latency, primary model of CD4+ T cells infected with HIV-1 as well as in ex vivo cultures of CD8+-depleted PBMCs from ART-treated HIV+ patients. CRISPR/Cas9-mediated depletion of ALKBH5 mimicked the effects of ALKi-3. ALKi-3 increased levels of m6A-methylated HIV-1 RNA as shown by meRIP and immuno-RNA FISH. Our study provides a proof-of-concept for the modulation of the m6A pathway in enhancing HIV-1 reactivation. This approach represents a promising adjunct to existing reactivation protocols and provides a concept of “dual-kick”, aiming to target transcriptional and post-transcriptional steps in HIV-1 reactivation from latency.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.