SARS-CoV-2 NSP8 suppresses type I and III IFN responses by modulating the RIG-I/MDA5, TRIF, and STING signaling pathways.

SARS-CoV-2 has developed a variety of approaches to counteract host innate antiviral immunity to facilitate its infection, replication and pathogenesis, but the molecular mechanisms that it employs are still not been fully understood. Here, we found that SARS-CoV-2 NSP8 inhibited the production of type I and III interferons (IFNs) by acting on RIG-I/MDA5 and the signaling molecules TRIF and STING. Overexpression of NSP8 downregulated the expression of type I and III IFNs stimulated by poly (I:C) transfection and infection with SeV and SARS-CoV-2. In addition, NSP8 impaired IFN expression triggered by overexpression of the signaling molecules RIG-I, MDA5, and MAVS, instead of TBK1 and IRF3-5D, an active form of IRF3. From a mechanistic view, NSP8 interacts with RIG-I and MDA5, and thereby prevents the assembly of the RIG-I/MDA5-MAVS signalosome, resulting in the impaired phosphorylation and nuclear translocation of IRF3. NSP8 also suppressed the TRIF- and STING- induced IFN expression by directly interacting with them. Moreover, ectopic expression of NSP8 promoted virus replications. Taken together, SARS-CoV-2 NSP8 suppresses type I and III IFN responses by disturbing the RIG-I/MDA5-MAVS complex formation and targeting TRIF and STING signaling transduction. These results provide new insights into the pathogenesis of COVID-19.

SARS-CoV-2 NSP5 and N protein counteract the RIG-I signaling pathway by suppressing the formation of stress granules.

As a highly pathogenic human coronavirus, SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread. The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity. However, whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive. In this study, SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule (avSG) formation. Moreover, NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA, poly (I:C), inhibiting TBK1 and IRF3 phosphorylation, and restraining the nuclear translocalization of IRF3. Furthermore, HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection. Mechanistically, NSP5 suppressed avSG formation and disrupted RIG-I-MAVS complex to attenuate the RIG-I-mediated antiviral immunity. In contrast to the multiple targets of NSP5, the N protein specifically targeted cofactors upstream of RIG-I. The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I. Additionally, the N protein also affected the recognition of dsRNA by RIG-I. This study revealed the intimate correlation between SARS-CoV-2, the stress response, and innate antiviral immunity, shedding light on the pathogenic mechanism of COVID-19.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5-MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.

The Interplay Between Pattern Recognition Receptors and Autophagy in Inflammation.

Pattern recognition receptors (PRRs) are sensors of exogenous and endogenous "danger" signals from pathogen-associated molecular patterns (PAMPs), and damage associated molecular patterns (DAMPs), while autophagy can respond to these signals to control homeostasis. Almost all PRRs can induce autophagy directly or indirectly. Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene-I-like receptors (RLRs), and cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway can induce autophagy directly through Beclin-1 or LC3-dependent pathway, while the interactions with the receptor for advanced glycation end products (RAGE)/high mobility group box 1 (HMGB1), CD91/Calreticulin, and TLRs/HSPs are achieved by protein, Ca2+, and mitochondrial homeostasis. Autophagy presents antigens to PRRs and helps to clean the pathogens. In addition, the induced autophagy can form a negative feedback regulation of PRRs-mediated inflammation in cell/disease-specific manner to maintain homeostasis and prevent excessive inflammation. Understanding the interaction between PRRs and autophagy in a specific disease will promote drug development for immunotherapy. Here, we focus on the interactions between PRRs and autophagy and how they affect the inflammatory response.