Biochemistry-informed design selects potent siRNAs against SARS-CoV-2

RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel Covid-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September 2022). Our approach is easily generalizable to other viruses as long as a variant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.

Low selectivity index of ivermectin and macrocyclic lactones on SARS-CoV2 replication in vitro argues against their therapeutic use for COVID-19.

There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC50 of 1-3 M). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC50 of 2-5 M). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se, due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.

A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

Genome-wide screens are powerful approaches to unravel new regulators of viral infections. Here, we used a CRISPR/Cas9 screen to reveal new HIV-1 inhibitors. This approach led us to identify the RNA helicase DDX42 as an intrinsic antiviral inhibitor. DDX42 was previously described as a non-processive helicase, able to bind RNA secondary structures such as G-quadruplexes, with no clearly defined function ascribed. Our data show that depletion of endogenous DDX42 significantly increased HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant increased infection. Importantly, DDX42 also restricted retrotransposition of LINE-1, infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 did not inhibit infection with three negative-strand RNA viruses, arguing against a general, unspecific effect on target cells, which was confirmed by RNA-seq analysis. DDX42 was found in the vicinity of viral elements by proximity ligation assays, and cross-linking RNA immunoprecipitation confirmed a specific interaction of DDX42 with RNAs from sensitive viruses. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results show for the first time a new and important role of DDX42 in intrinsic antiviral immunity.

Direct visualization of native infectious SARS-CoV-2 ant its inactivation forms using high resolution Atomic Force Microscopy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID19, a new emerging pandemic affecting humans. Here, single viruses were analyze by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess infectious virus morphology in its native conformation, or upon inactivation treatments, at the nanoscale level and in 3D. High resolution AFM reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory, as revealed by combining AFM and plaque assays, to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-atomic force microscopy is a remarkable toolbox for rapid and direct virus identification and characterization.