| Porcine epidemic diarrhea virus(PEDV)is a pathogen that causes Porcine epidemic diarrhea(PED).PEDV infection in piglets can cause severe diarrhea and dehydration,and the fatality rate is very high.Over the past few decades,PEDV poses a great threat to the global pig industry with the continuous evolution and spread of the virus,which has brought significant economic losses.The type I interferon response,as the first line of defense and the main component of the host cell’s natural immune response,can restrict the life activity of the virus at the initial stage of infection,and the main purpose of the virus is to escape or antagonize the interferon response.PEDV has evolved a variety of mechanisms to evade or counter the host immune response to ensure its normal life activities.PEDV Nsp14 protein is one of the key proteins,and Nsp10 protein is often used as an auxiliary protein of Nsp14 protein to enhance its activity in coronavirus.It has been documented that PEDV Nsp14 protein can inhibit host cell type I interferon response,but its specific mechanism has not been fully revealed.This paper aims to explore the detailed mechanism of PEDV Nsp14 protein and Nsp10 protein coinhibiting host cell type I interferon response from the following two aspects:(1)Explore the influence of PEDV Nsp14 protein on the signaling pathway of host type I interferon response,and the role of the enzyme activity of Nsp14 protein in inhibiting host cell type I interferon response,and clarify the molecular mechanism of its inhibition of host type I interferon response.(2)Explore and clarify the influence of PEDV Nsp10 protein interaction with Nsp14 protein on the response of host type I interferon and its molecular mechanism.In this study,Western blot and RT-PCR were used to detect the changes of key proteins of type I interferon signaling pathway during the overexpression of Nsp14 protein in IPEC-J2 cells.The methylase activity of coronavirus plays an important role in regulating host innate immunity,and PEDV Nsp14 protein has an N7 methyltransferase activity region.Therefore,in this study,after overexpression of Nsp14 protein with N7 methyltransferase activity deletion in IPEC-J2 cells,its influence on key proteins of type I interferon signaling pathway was detected,so as to explore the role of Nsp14 protein and its N7 methyltransferase activity in regulating the response of host cells with type I interferon.Then,in this study,PEDV Nsp10 recombinant plasmid was constructed using molecular cloning technology,and site-specific mutation technology was used to construct residual mutant Nsp10 mutant plasmid interacting with Nsp14 protein.Subsequently,PEDV Nsp14 protein,Nsp10 protein and Nsp10 mutant protein were co-transfected into host cells,and the interaction between the two proteins in host cells was detected by Co-IP experiment,Western blot and RT-PCR.And the changes of key proteins of type I interferon signaling pathway in host cells to explore the molecular mechanism of PEDV Nsp10 protein and Nsp14 protein’s synergic action on type I interferon response in host cells.Experimental results show that PEDV Nsp14 can inhibit the phosphorylation levels of IRF3 protein and STAT1 protein and the transcription levels of IFN-β and ISGs in host cells.Moreover,when the N7 methyltransferase activity of PEDV Nsp14 is absent,The inhibitory effect of Nsp14 protein on host cell IRF3/IFN-β/STAT1/ISGs,a type I interferon response signaling pathway,disappeared.When Nsp14 was present with Nsp10,it had a stronger ability to inhibit the type I interferon response signaling pathway in host cells,and Nsp10 had no effect on the signaling pathway.PEDV Nsp14 protein interacts with Nsp10 protein in host cells.Compared with wild-type Nsp10 protein control group,when the interaction between Nsp10 mutant protein(K43A,H80A)and Nsp14 protein is reduced or disappeared,The ability of Nsp14 to inhibit host cell type I interferon signaling pathways enhanced by Nsp10 protein is also reduced or eliminated.In this study,PEDV Nsp14 protein has the function of inhibiting host cell type I interferon response,and this function depends on its N7 methyltransferase activity.PEDV Nsp10 protein can enhance the inhibitory effect of Nsp14 protein on type I interferon,and the effect of Nsp10 protein depends on its interaction with Nsp14 protein.This study elucidates the molecular mechanism by which Nsp14 and Nsp10 cooperate to inhibit the response of host cell type I interferon.Given the conserved nature of Nsp14 and Nsp10 proteins in coronavirus,this study can also provide theoretical basis for the design of effective drugs for the treatment of other coronavirus infections. |
