Molecular Mechanism Of Porcine Epidemic Diarrhea Virus Membrane Protein To Inhibit Type ? Interferon Production

Porcine epidemic diarrhea virus(PEDV)is a highly pathogenic porcine enteropathogenic coronavirus,belonging to the genus Alphacoronavirus,causing severe enteritis and lethal watery diarrhea in piglets.The mortality rate was 100% in the most serious epidemics,especially in 2010,the emergence of PEDV highly pathogenic strains caused PED outbreaks in several countries successively,which result in serious damage to the global pig industry and biological safety.Virus invasion induces the host antiviral innate immune response.Type ? interferon is the main antiviral cytokine in the innate immune defenses,and has powerful effect of inhibiting virus replication.In the process of virus interacting with the host immune system,coronaviruses have evolved a series of antagonistic mechanisms to inhibit innate immunity for promoting virus replication.The molecular mechanisms by which ? and ? coronaviruses escape from innate immune responses have been widely studied.Although,some studies indicate that PEDV infection suppresses the synthesis of type ? interferon(IFN).Of 23 viral protein of PEDV,11 proteins were identified as interferon antagonists,and multiple viral proteins of PEDV have been shown to target the adaptors of RIG-I pathway to inhibit type ? IFN production.PEDV induced immunosuppressive mechanisms are still not fully established.Coronavirus M proteins play important roles in viral assembly,membrane fusion,and induction of neutralizing antibody production,and have been shown to antagonize IFN production by targeting key adaptor molecules of innate immunity signaling.To better reveal the immunosuppressive mechanism of PEDV,the antagonistic property of IFN of nsp14,nsp15,nsp16 and M protein were evaluated,and the molecular mechanism of M protein to inhibit IFN was clarified.Here,we identified PEDV membrane(M)protein as a new antagonist of type ? IFN production and demonstrated that M inhibited the activation of RIG-I-,TLR3-and TLR7 signaling pathways.Further studies determined the antagonistic mechanism by M protein to target interferon regulatory factor 7(IRF7),an important regulator of type ? IFN production.IRF7 is phosphorylated and activated by TBK1 and IKK? in response to viral infection.We found that PEDV M protein interacted with the ID domain of IRF7 and significantly suppressed TBK1/IKK?-induced IRF7 phosphorylation and dimerization of IRF7,leading to the decreased expression of type ? IFN.As expected,overexpression of M protein significantly increased PEDV replication in porcine cells.The M proteins of both field PEDV strains and vaccine strain showed similar antagonistic effect on type ? IFN production,and the 1-55 region of M protein was determined to be essential for disruption of IRF7 function.Taken together,PEDV M protein inhibited the activation of RIG-I-,TLR3-and TLR7 signaling pathways.M protein suppressed activation of IRF7 by interacting with the ID domain of IRF7,leading to the reduced expression of IFN-I and its downstream antiviral genes,and then promoted PEDV replication.The 1-55 amino acid region of M protein was essential for inhibiting IRF7 function.Our data identified a novel antagonistic mechanism evolved by PEDV to inhibit type ? IFN production.It provides a new theoretical basis for elucidating the pathogenic mechanism of PEDV and the development of effective vaccine for PEDV.