Molecular Basis Of The Effects Of Different Sites Patterns Of NS1 Protein Effector Domain Of H1N1 Subtype Influenza Virus On IFN-β And Apoptosis

H1N1 is the most important subtype of influenza A virus infecting mammals.It causes upper and lower respiratory tract infections in animals,resulting in clinical symptoms such as fever,cough,anorexia and lethargy,which affect the growth and development of animals and cause severe mortality.It not only brings huge economic losses to the farming industry,but also threatens global public health security.Non-structural protein 1（NS1）,a protein of influenza virus,exists only in infected cells but not in viral particles.It plays an important role in regulating host gene expression,antagonizing interferon（IFN）to inhibit host innate immune response,regulating apoptosis and affecting virus virulence.In this study,it was found that there were significant differences in amino acids at the 90,123,125,131 and 205 sites between the strains prevalent before 2009 and the strains prevalent in 2009 and later by comparing the H1N1 NS gene sequences in the Geen Bank and GISAID datebases Accordingly,six different NS1 patterns were constructed then linked to the eukaryotic expression vector p EGFP-N1,respectively.Subsequently,each virus was rescued by reverse genetic techniques to detect the influence of NS1 proteins on IFN-βand apoptosis.The results of immunofluorescence and Western blot experiments showed that each p EGFP-NS1 was able to express and localize normally.After the addition of interferon inducer,the NS1 proteins expression were inhibited,and the p EGFP-NS1/P6 was more significantly inhibited compared with the p EGFP-NS1/P1.On this basis,we investigated the influence of NS1 proteins on IFN-βand its related genes.The results of dual luciferase assay showed that the NF-κB and IRF3promoter activities were higher in p EGFP-NS1/P2 than others,while they were lower in p EGFP-NS1/P5 and p EGFP-NS1/P6.The q PCR results showed that compared with p EGFP-NS1/P1,p EGFP-NS1/P2 had significantly higher transcript levels of RIG-I,IFN-βand ISGs,while pEGFP-NS1/P5 and pEGFP-NS1/P6 had significantly lower transcript levels of RIG-I,IFN-β,Mx and ISG15.ELISA experiments showed that compared to p EGFP-NS1/P1,p EGFP-NS1/P2 showed significantly higher expression of IFN-βand ISG15 in both cells and cell supernatants at 24 h and 48 h,while the pEGFP-NS1/P6 was significantly lower.Then we examined the influence of NS1 proteins on the IFN-βexpression under apoptosis induction.As shown by q PCR and ELISA,the transcription and expression of IFN-βand ISG15 were significantly increased in p EGFP-NS1/P2 whileas significantly decreased in p EGFP-NS1/P6 compared with p EGFP-NS1/P1.Subsequently,the influence of NS1 proteins on apoptosis under interferon induction were examined.As shown by qPCR and Western blot,the transcription and expression of the pro-apoptotic proteins,Bax and caspase-3,were significantly higher in p EGFP-NS1/P2 compared with p EGFP-NS1/P1.Whereas the transcription of the anti-apoptotic protein,Bcl-2,was significantly lower in p EGFP-NS1/P5 and p EGFP-NS1/P6 compared with p EGFP-NS1/P1.The caspsae-3 activity assay results showed that compared to p EGFP-NS1/P1,the activity was significantly higher in p EGFP-NS1/P2,while it was significantly lower in p EGFP-NS1/P5 and p EGFP-NS1/P6.After successfully rescuing the virus,the growth curves of NS1 viruses were similar as measured though TCID₅₀.But the r NS1/P6 had stronger viral proliferation than others.The q PCR and subsequent apoptosis experiments yielded similar results to the plasmid level.The results of q PCR and caspase-3 activity assays to detect the effects of NS1 viruses on IFN-βand apoptosis were similar to the plasmid level.In summary,the results of plasmid and virus levels showed that the ability of NS1-P6 antagonized IFN-βand regulated apoptosis was stronger than other viruses,while NS1-P1/P2 was relatively weaker.In this study,we focused on investigating the differences of variant NS1 protein amino acid patterns on antagonizing IFN-βand regulating apoptosis by constructing six different NS1,so as to reveal the molecular basis of IFN and apoptosis regulation by NS1 ED region.