Effect Of NS1 Site-directed Mutation Of H9N2 Subtype Influenza Virus On The Transcription Of Type-? Interferon And ISGs

H9N2 subtype,as one kind of low pathogenic influenza A virus,has been spread widely in more than 20 provinces after the first isolation in 1992 in mainland China.It is also a main subtype of influenza A virus,which has been harming the poultry industry of China.Now,the infection spectrum of this subtype is widespread and is still widening,which is a great threat to the public health.As the only non structural protein of influenza A virus,NS1 protein plays an important role in antagonizing the innate immune response,so that influenza A virus can effectively proliferate and replicate in the host cells.After blasting the amino acid sequences of NS1 proteins of all 2,237 different strains of H9N2 subtype downloaded from Gen Bank,it was found that 114S accounted for 96.02%,114P only accounted for 1.25%;124V accounted for 54.55%,124 M accounted for 42.56%;202A accounted for 95.10%,202 T only accounted for 4.60%;212P accounted for82.94%,202 S just accounted for 15.51%.The amino acid residues at position 114,124,202,212 of NS1 protein of G1 strain used in this study was P(2.03%),M(42.56%),T(13.17%)and S(6.37%)respectively.And it was said that the amino acid differences may affect the ability of antagonizing innate immune response of NS1 protein.Therefore,it is necessary to study the effect of these 4 amino acid sites on the function of NS1 protein of Influenza A virus.In this study,the amino acid residues at positions 114,124,202 and 212 in NS1 protein of A/Quail/Hong Kong/G1/1997(H9N2)were substituted by the overlapping PCR,then the site-directed recombinant expression plasmids p CAGGS-NS1/P114S,p CAGGS-NS1/M124V,p CAGGS-NS1/T202A,p CAGGS-NS1/S212P and non-mutant recombinant expression plasmid p CAGGS-NS1 were constructed.According to the result of dual-luciferase reporter assay,compared with poly(I:C)treatment,NS1 protein without mutation and NS1 protein with P114S,M124V or S212P mutation can all inhibit the transcription activity of NF-?B,IRF-3 and IFN-?promoter,but NS1 protein with T202A mutation has no significant inhibitory effect on NF-?B,IRF-3 and IFN-? promoter.Compared with the treatment of NS1 protein without mutation,NS1 protein with S212P mutation has the most significant inhibitory effect on transcription activity of NF-?B,IRF-3 and IFN-? promoter,while the inhibition level of transcription activity of NF-?B,IRF-3 and IFN-?promoter was obviously decreased when NS1 protein with T202A mutation.This result showed that,compared with the NS1 protein without mutation,NS1 protein with P114S,M124V or S212P mutation may further inhibit the function of IFN-?and its pathway molecules,but the NS1 protein with T202A mutation may not inhibit the function of IFN-? and its pathway molecules any further.The site-directed recombinat viruses ReG1-NS1/P114S,ReG1-NS1/M124V,ReG1-NS1/T202A,ReG1-NS1/S212P and non-mutant ReG1-NS1 were also rescued by the reverse genetic technique.The TCID50 values of ReG1-NS1/P114S,ReG1-NS1/M124V and ReG1-NS1/S212P were clearly less than the values of ReG1-NS1 and ReG1-NS1/T202A,which may be due to NS1 protein with P114S,M124V or S212P mutation increasely inhibited the function of IFN-? and its pathway molecules.So that these 3 recombinant strains can replicate and proliferate better in host cells.The transcription level of IFN-? and ISGs mRNA in A549 cells after 9h post infection showed that,compared with ReG1 treatment,the transcription level of IFN-? and ISGs mRNA were inhibited further after infected by ReG1-NS1/P114S,ReG1-NS1/M124V or ReG1-NS1/S212P.And the inhibitory effect of ReG1-NS1/S212P on the transcription level of IFN-? and interferon stimulating gene mRNA was the most significant.But compared with ReG1 treatment,the transcription level of IFN-? and ISGs mRNA were not significantly changed after ReG1-NS1/T202A infection.The transcription level of IFN-?,ISGs and inflammatory factors mRNA in the lungs of Chicken after 3 and 7 days post challenge showed that,compared with ReG1 treatment,the transcription level of the pathway molecules of type I interferon and inflammatory factors mRNA could be further decreased after ReG1-NS1/P114S,ReG1-NS1/M124V or ReG1-NS1/S212P challenged.And the inhibitory effect of ReG1-NS1/S212P on the transcription level of the pathway molecules of type I interferon and inflammatory factors mRNA was the most significant.But compared with ReG1 treatment,the transcription level of the pathway molecules of type I interferon and inflammatory factors mRNA were also not significantly changed after ReG1-NS1/T202A challenged.The results above displayed effects of NS1 mutation of H9N2 subtype influenza virus on biological characteristics and expression of IFN-? in both protein and virus level.Compared with the non mutated NS1 protein,NS1 protein with P114S,M124V or S212P mutation,can further inhibit the transcription level of type I interferon,ISGs and even inflammatory factors mRNA,so as to antagonizing host innate immune response and inflammatory reaction.But NS1 protein with T202A mutation has no further inhibitory effect on the transcription level of type I interferon,ISGs and inflammatory factors mRNA.