The RNA helicase DHX16 recognizes specific viral RNA to trigger RIG-I-dependent innate antiviral immunity.

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.

Polyphenylene carboxymethylene (PPCM) microbicide repurposed as antiviral against SARS-CoV-2. Proof of concept in primary human undifferentiated epithelial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected over 200 million people throughout the world as of August 2021. There are currently no approved treatments providing high chance of recovery from a severe case of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, and the beneficial effect of Remdesivir and passive immunization therapies may only be seen when administered early on disease onset. The emergence of variants is also raising concerns regarding the efficacy of antibody therapies, antivirals, and vaccines. Therefore, there is still a need to develop new antivirals. Here, we investigated the suitability of primary human epithelial cells from the trachea/bronchia (NHBE) and small airway (SAEC) as lung models of SARS-CoV-2 infection to determine, whether the microbicide polyphenylene carboxymethylene (PPCM) has antiviral activity against SARS-CoV-2. Both NHBE and SAEC expressed proteins required for virus entry in lung epithelial cells. However, these cells were only low to moderately permissive to SARS-CoV-2 as titers increased at best by 2.5 log10 during an 8-day kinetic. Levels of replication in SAEC, unlike in NHBE, were consistent with data from other studies using human normal tissues or air-liquid interface cultures, suggesting that SAEC may be more relevant to use than NHBE for drug screening. PPCM EC50 against SARS-CoV-2 was between 32 and 132 µg/ml with a selectivity index between 12 and 41, depending on the cell type and the infective dose used. PPCM doses were consistent with those previously showing effect against other human viruses. Finally, PPCM antiviral effect observed in SAEC was in line with reduction of inflammatory markers observed overly expressed in severe COVID-19 patients. Altogether, our data support the fact that PPCM should be further evaluated in vivo for toxicity and antiviral activity against SARS-CoV-2.

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Spike mutation D614G alters SARS-CoV-2 fitness.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.