Studies On The Molecular Mechanism Of Porcine Epidemic Diarrhoea Virus Non-structural Protein Nsp7 Inhibiting IFN-? Signaling Inhibited Type ? Interferon Signaling

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus(PEDV)has caused huge economic losses to the world's pig industry.PEDV belongs to the Nidovirales,coronaviridae family.The genome size is about 28 kb,and the genome encodes four structural proteins(E,M,S,N),16 non-structural proteins(nsp1-nsp16)and one accessory protein ORF3.In the process of virus infection,the host cells can produce a variety of molecules to resist the invasion of the virus,including interferon(IFN),pro-inflammatory cytokines and chemokines.Among them,Interferon and its induced interferon-stimulating genes(ISGs)are important defense mechanisms for host antiviral infections,but viruses often evolve strategies to antagonize the antiviral activity of host IFN to achieve the purpose of infecting the host.Studies have confirmed that PEDV has the ability to inhibit type I IFN production and its signal transduction,but its encoded nsp7 protein in which the role is not clear.In view of this,this paper takes PEDV nsp7 as the object of study,the main contents are as follows:1.PEDV nsp7 inhibits the activation of ISRE and production of ISGsPEDV can infect pigs of different ages,so it is worthy of our attention to study the strategy of immune escape.And many viruses antagonize IFN immune response is mostly by virtue of its encoded protein to achieve.Therefore,this study screened the PEDV encoding protein involved in the inhibition of ISRE promoter activation.The results showed that nsp7 could significantly inhibit the activity of IFN-?-mediated ISRE promoter,and its inhibitory effect was dose-dependent.At the same time,the Real-Time PCR showed that PEDV nsp7 could significantly inhibit the expression of ISG15,ISG54 and ISG56 at m RNA level.2.PEDV nsp7 does not inhibit the phosphorylation of JAK1,Tyk2,STAT1,STAT2 and formation of ISGF3During the evolution,virus have formed a variety of strategies to disrupt the ?-IFN signal transduction.Western blot analysis showed that PEDV nsp7 did not affect the protein expression and phosphorylation of JAK1,Tyk2,STAT1 and STAT2,nor did ISGF3 complex formation be affected,but can significantly inhibit ISGF3-induced ISRE promoter activation and interact with ISGF3.The nuclear translocation of ISGF3 is a necessary part of ISGs generation.PEDV nsp7 does not affect STAT1,STAT2 expression and phosphorylation and ISGF3 complex formation,but inhibit ISGF3-induced ISRE promoter activity,then will it affect STAT1,STAT2 transfer to the nucleus? We performed indirect immunofluorescence assay and the cytoplasmic and nuclear extraction experiments respectively.The resultsshowed that PEDV nsp7 could significantly inhibit the nuclear translocation of STAT1,STAT2 and IRF9.3.PEDV nsp7 competes with KPNA1 in combination with STAT1,blocking ISGF3 nuclear transportSince PEDV nsp7 significantly inhibited the translocation of STAT1,STAT2 and IRF9 and interacted with ISGF3 complexes,it was presumed that PEDV nsp7 might interact with STAT1,STAT2 or IRF9.Through immunoprecipitation experiments we conclude that there is no interaction between PEDV nsp7 and IRF9,but PEDV nsp7 can interact with STAT1 and STAT2,and specifically binds to the DBD domain of STAT2.The DBD is highly homologous in the STATs protein family and has atypical NLS sequence,which are key amino acid sequences identified by KPNA1 to the ISGF3 complex.In view of this,we use CO-IP experiment to detect whether nsp7 affects the binding of KPNA1 to STAT1.The results showed that the interaction between KPNA1 and STAT1 gradually decreased with the increase of nsp7 expression.Therefore,nsp7 could inhibit the combination of STAT1 and KPNA1 by targeting STAT1,which specifically blocked the entry of ISGF3 into the nucleus.4.Disruption of ?-IFN signal pathway by PEDV nsp7 has nothing to do with nsp8.Studies have shown that the feline coronavirus the nsp7 / nsp8 complex has a 2: 1 heteromeric 3-mer structure,and the binding to nsp7 can enhance the RNA polymerase activity of nsp8.The nsp7 / nsp8 of SARS coronavirus form hexadecameric ring structure in 8: 8,Which can significantly enhance the Rd Rp activity possessed by nsp8 and have primer extension ability.So,is the inhibition of JAK-STAT signaling pathway caused by nsp7 associated with nsp8? We transfected nsp7 and nsp8 separately or co-transfected with different proportions into HEK-293 T cells,and analyzed by dual luciferase reporter system.The results showed that nsp8 does not correspond to ISRE promoter activation,and the inhibitory effect of nsp7 on the ISRE promoter activity was not dependent on nsp8.