Mechanisms Of SARS-CoV-2 NSP5 And N Suppressing Host Antiviral Immune Responses

The unexplained pneumonia that broke out in December 2019 has received extensive attention from all walks of life because of its high infectiousness and serious threat to human health and safety.After the researchers isolated the virus-causing strain from the patient,the virus structure was further analyzed,and the genome sequence was determined to confirm that the virus belongs to the coronavirus family.Compared with two other coronaviruses(SARS-CoV,MERS-CoV)that can infect humans,the novel coronavirus(SARS-CoV-2,2019-nCoV)is more contagious and can spread rapidly among the population,leading to an acute and rapidly developing global health crisis.As a highly pathogenic RNA virus,the novel coronavirus needs to antagonize the complex antiviral immune response signaling network in the host to achieve its infection and spread in the population.And in existing studies,the analysis of samples from COVID-19 patients,it was found that compared with other respiratory virus infections,2019-nCoV infection showed lower secretion of type Ⅰ andⅢ interferon,and higher secretion of inflammatory cytokines,which indicates that SARS-CoV-2 can effectively regulate the host’s innate antiviral immune system.As an RNA virus,SARS-CoV-2 is mainly recognized by the host’s RLRs and TLRs signaling pathways,thereby affecting the host’s innate antiviral immune system.Moreover,when viral infection occurs in the host,the viral nucleic acid released into the host can effectively induce the formation of stress granules(SGs),and the production of SGs further inhibits the translation of viral mRNA,thereby blocking the replication and reproduction of the virus.Therefore,Stress granules play an important role in the body’s resistance to viral infection and are closely related to the antiviral innate immunity of the host.Existing studies have found that various proteins encoded by SARS-CoV-2 can affect the natural antiviral immune network in the host through different ways,and some of them can exert their immunosuppressive functions through a variety of different mechanisms.However,it remains unknown whether the protein encoded by SARS-CoV-2 can achieve immune escape by regulating the body’s stress response.It has been reported in the literature that plasmid transfection can effectively induce the formation of stress granules.The plasmids expressing viral proteins can be transfected into cells,and then whether each viral protein affects the formation of stress granules can be observed by immunofluorescence.Therefore,in this experiment,the related genes that can affect the formation of stress granules in the proteins encoded by SARS-CoV-2 were firstly screened by using this experimental method.The effects of these two genes on the innate immune response during SARS-CoV-2 infection were then further investigated.In this study,we transfected the expression vectors of various proteins encoded by SARS-CoV-2 into HeLa cells,and then observed the effects of these proteins encoded by SARS-CoV-2 on the formation of stress granules by immunofluorescence technology.The results showed that when cells were transfected with the expression vector of SARS-CoV-2 NSP5 or N,the formation of stress granules in cells could be significantly inhibited.Afterwards,the roles of NSP5 and N in virus infection were detected by fluorescence microscopy,flow cytometry and plaque assay,and it was found that the expression of NSP5 or N could effectively promote virus replication.Then,we also tested whether the expression of NSP5 and N affects the synthesis of downstream effector molecules by qRT-PCR.The results showed that the expression of NSP5 or N protein effectively inhibited the expression of IFN-β,IFN-λ1,ISG56 and CXCL10 induced by SeV and poly(I:C)stimulation.At the same time,the expression of NSP5 or N can significantly inhibit the phosphorylation of downstream signaling molecules TBK1 and IRF3 in the RLRs signaling pathway,as well as the translocation of IRF3 into the nucleus,by Western Blot experiments.Further mechanism studies found that both NSP5 and N inhibited the body’s antiviral immunity through multiple pathways.We detect by Western Blot and immunofluorescence experiments,it was found that both NSP5 and N could block the formation of stress granules by interacting with G3BP1.At the same time,we co-transfected NSP5 or N with each linker molecule in the RLRs signaling pathway,and then detected by co-immunoprecipitation or immunofluorescence.The results showed that both NSP5 and N interacted with RIG-1 and MDA5.Then,the double-luciferase reporter gene method was used to detect that the expression of NSP5 could effectively reduce the activation of RIG-1 and MDA5-mediated signaling pathways,while the expression of N did not affect the activation of the signaling pathway induced by any linker molecule,this result reminds us that NSP5 may play a role in the upstream and downstream of RIG-I,and the target of N may be located upstream of RIG-I.Then we detected by co-immunoprecipitation and found that the expression of NSP5 can effectively reduce the interaction between RIG-I with MAVS,TRIM25 with RIG-I,and then affect the host’s antiviral immune process.Then,through further research,we found that the N protein can firstly interact with PACT,the cofactor of the RLRs signaling pathway,inhibiting the enhancing effect of PACT on the activation of this signaling pathway.In addition,it also inhibits the body’s antiviral immune response by affecting the interaction between TRIM25 and RIG-I.Finally,because N contains two RNA-binding motifs from a structural point of view,through further experiments,we found that the expression of N can significantly inhibit the ability of RIG-I to recognize RNA,thereby affecting the activation of downstream signaling pathways.In conclusion,NSP5 and N not only affects the formation of stress granules,but also affects the activation of the signaling pathway,by affecting different stages of the RLRs signaling pathway.In turn,it inhibits the phosphorylation of downstream signaling molecules in the signaling pathway and the nuclear translocation of IRF3,thereby inhibiting the secretion of interferon.In this study,we found that the NSP5 and N proteins encoded by SARS-CoV-2 can obviously inhibit the formation of antiviral stress granules(avSGs)and promote virus replication.Then,the specific mechanism of N or NSP5 inhibiting the body’s antiviral immunity was further elucidated.This study reveals the interrelationship between SARS-CoV-2,stress response and antiviral innate immunity,which helps to further reveal the pathogenic mechanism of COVID-19.Innovation:1.This study proposes for the first time that the NSP5 and N protein of SARS-Cov-2 can effectively inhibit the host’s antiviral ability by affecting the formation of stress granules.2.The effect of NSP5 on the formation of stress granules does not depend on its cleavage enzyme activity.At the same time,NSP5 can also inhibit its activation by affecting multiple steps in the RLRs signaling pathway.3.The protein N encoded by SARS-CoV-2 inhibits the activation of the signaling pathway induced by the co-factors(PACT,G3BP1)of the RLRs recognition receptors through regulating the formation of SGs.