SARS-CoV-2 NSP8 Suppresses IFN Responses By Modulating The RIG-Ⅰ/MDA5,TRIF,and STING Signaling Pathways

COVID-19(Corona Virus Disease 2019)is a kind of acute respiratory disease caused by SARS-CoV-2(severe acute respiratory syndrome coronavirus 2)that has spread rapidly,threatened public health seriously and restricted the development of the global economy.SARS-CoV-2 belongs to the β-coronavirus genus of Coronaviridae family and its genome is linear positive single-stranded RNA,which is the largest RNA virus.The genome of SARS-CoV-2 encodes multiple proteins,including 16 non-structural proteins,4 structural proteins and several accessory proteins.Studies has shown that some non-structural proteins of SARS-CoV-2 are also involved in the inhibition of host innate immunity.Innate immunity is the first line of defense against foreign invasion for the host.Double-stranded RNA(dsRNA)generated during the replication of SARS-CoV-2 can activate effectively pattern recognition receptors(PRRs)in the innate immune response,such as Toll-like receptor 3(TLR3)located on endosomes,retinoic acid-inducible gene Ⅰ(RIG-Ⅱ)and melanoma differentiation associated factor 5(MDA5)located in the cytoplasm.In addition,SARS-CoV-2 infection also leads to the release of mitochondrial DNA and chromatin DNA,which activate the cGAS-STING pathway that recognizes DNA in the cytoplasm.After binding with their corresponding ligands dsRNA or DNA,TLR3,RIG-I/MDA5,and cGAS will activate downstream adaptor proteins TRIF,MAVS,STING,respectively;and these three signaling pathways converge at tank binding kinase 1(TBK1)to phosphorylate transcription factor interferon regulatory factor 3(IRF3),thus promoting the expression of type Ⅰ and Ⅲ interferons(IFNs).Type Ⅰ and Ⅲ IFNs further initiate the Janus tyrosine kinase(JAK)/signal transducer and activator of transcription(STAT)signaling pathway via autocrine or paracrine pathways,inducing the expression of interferon-stimulated genes(ISGs)and promoting the host cells establish an antiviral immune state rapidly.A typical feature of SARS-CoV-2 infection is the suppression of antiviral immunity response and over-activation of the inflammatory response.Clinical studies have shown that treatment with type Ⅰ interferon can effectively alleviate the symptoms of COVID-19 patients.This suggests that SARS-CoV-2 have a unique innate immune evasion mechanism.Therefore,exploring the innate immune evasion mechanism of SARS-CoV-2 is of great significance for the treatment of COVID-19.In this study,we performed RT-qPCR assays and dual-luciferase reporter systems,by which we found that SARS-CoV-2 NSP8 protein can inhibit the IFN response induced by Sendai virus(SeV)or the dsRNA analog poly(I:C).Flow cytometry and virus plaque assays revealed that overexpression of NSP8 can promote virus replication and proliferation.Further experiments showed that NSP8 can inhibit the RIG-Ⅰ/MDA5-MAVS,TLR3-TRIF,and cGAS-STING signaling pathways.Mechanistic studies revealed that NSP8 interacts with RIG-Ⅰ,MDA5,TRIF,and STING,and overexpression of NSP8 can reduce the binding between MDA5-MAVS,TRIF-TBK1,and STING-TBK1.In addition,NSP8 reduces the phosphorylation and nuclear translocation of IRF3,thus inhibiting IFN expression and downstream JAK/STAT signaling pathway activation.In summary,this study shows that SARS-CoV-2 NSP8 protein targets RIG-Ⅰ/MDA5-MAVS,TLR3-TRIF and cGAS-STING signaling pathways to inhibit the production of type Ⅰ and Ⅲ IFNs and the activation of antiviral immunity,it contributes to our understanding of the molecular mechanism through which SARS-CoV-2 escapes host innate antiviral immunity,thereby providing an essential clue to the pathogenesis and a theoretical basis for drug development of COVID-19.