Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy

SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spikes polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

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.

SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.