The Mechanism Study Of SADS-CoV NS7a On Regulating Type ? Interferon Pathway

Accompanied by genetic evolution,the organism gradually develops an innate immune defense ability to prevent the invasion of foreign microorganisms or pathogens.This innate immune system is called natural immunity.As the body's first defensive line against foreign pathogens,it characterizes as non-specificity,broad-spectrum,rapid response,and heredity.The type ? Interferon(IFN-I)signaling pathway is an essential part of the natural immune process.When pathogens and microorganisms invade the cells of the body,intracellular pattern recognition receptors can recognize the pathogens and their products and mediated downstream signaling pathway activation through a cascade reaction.The signal pathway generates a series of IFN-stimulated genes(ISGs)to produce interferons,which directly or indirectly mediate the clearance of pathogens and maintain immune balance.Coronavirus(CoVs)are widespread,with a broad host range,and commonly infect humans,mammals,and poultry.In recent years,the successive outbreaks of SARS-CoV,MERS-CoV,and the novel coronavirus in 2019 have severely affected the health of human life and the regular orders of social and economic development,causing people to pay strong attention to the coronavirus.In the pig industry,TGEV,PEDV,PDCoV,and SADS-CoV also caused severe damage in pig farms.Thus,the investigations to reveal the mechanism behind the rapid transmission ability and the strong lethality of the coronavirus are urgently needed.In this study,the NS7 a protein of SADS-CoV was used as the target to study its role and potential regulatory mechanism in the response of the type ? interferon signaling pathway.The dual-fluorescence reporter gene system was used in this study to evaluate the influences of NS7 a on the promoter activity of IFN?,ISRE,IRF3,and NF-?B induced by Se V and poly(I:C).The m RNA levels of IFN? and ISGs were also detected by real-time PCR in HEK293 T cells.The effect of NS7 a on the protein expression level of signal molecules in signaling pathway induced by poly(I:C)was measured by protein immunoblotting.The results showed that NS7 a significantly inhibited the promoter levels of IFN?,ISRE,and NF-?B.At the m RNA level,NS7 a has a significant inhibitory effect on the m RNA transcription levels of DDX58,MDA5,IRF3,TBK1,STING,STAT2,OASL,ISG15,and IFIT2.Similarly,NS7 a significant downregulated the protein levels of TBK1,P-TBK1,STING,P-STING,IRF3,P-IRF3,STAT1,P-STAT1,P-STAT2,and IRF9.According to the trend shown by the previous data,this study first constructed the eukaryotic expression plasmids of each molecule of the IFN-I pathway to find the possible target of NS7 a.Combined the previous data,the upstream molecules of the signaling pathway were focused to identify their interactions with NS7 a protein.Unfortunately,the co-IP results showed that NS7 a did not directly interact with the upstream molecules of the IFN-I pathway.However,abnormal cell morphology and increased dead cells were observed when the HEK293 T cells were transfected with NS7 a,suggesting cell apoptosis may be induced by NS7 a.Using microscopic morphological observation,protein immunoblotting and flow cytometry and other classic apoptosis detection methods,we discovered that NS7 a not only strongly inhibited the interferon signal pathway,but also strikingly induced the apoptosis of HEK293 T cells at48 hours post transfection.After 44 hours upon NS7 a transfection of cells,late apoptosis accounted for about 12%,and early apoptosis accounted for approximate 40%.The endogenous co-IP results showed that NS7 a interacted with caspase-8 zymogen.Further investigations revealed that Z-VAD was unable to inhibit HEK293 T cell apoptosis induced by NS7 a.In summary,this study found that NS7 a of SADS-CoV inhibited the poly(I:C)-activated type ? interferon signaling pathway and induced apoptosis in HEK293 T cells.We postulate that NS7 a interacts with caspase-8 to activate apoptosis via the extrinsic pathway and causes apoptosis of HEK293 T cells.