Effect Of S212P And I178V Mutations On The Function Of H7N9 NS1 Protein

Since early 2013,zoonotic transmission of novel avian-origin H7N9 influenza A virus has posed a great threat to human health and caused social panic.NS1 is a multifunctional protein of influenza virus and helps the virus to evade the innate immune response of host.Previously,the function of H7N9 NS1 protein has been studied in this laboratory and it was found that mutations in S212 and 1178 could affect the function of NS1.However,the precise role of the above two sites of NS1 protein in antagonizing the host antiviral response is not fully understood.In this study we performed detailed experiments to analyze the functions of NS1 during influenza A virus infection following its mutation on S212 and 1178.In this study,the eukaryotic expression plasmid of H7N9 NS1 protein and plasmid carrying mutations(E70K,T143A,S212P)were transfected into 293T cells for 24 hours.After infection with influenza A/WSN/33(H1N1),RT-PCR was performed to examine the effect of mutation of S212 amino acid site on the host innate immune response.The H7N9 NS1 protein and its S212P mutant plasmid were transfected into 293T cells for 24h.Transfected cells were then infected with A/WSN/33(H1N1)and A/PR/8/34(H1N1)ΔNS1 influenza virus strains respectively.The mRNA expression levels of RIG-I,MDA5,TLR3 and ISGs were detected by RT-PCR and real-time PCR and the phosphorylation levels of STAT1 protein were examined by Western blotting.Finally,Western blotting and plaque assay were used to detect the replication of influenza virus.The results are shown as follows:1)Compared with NS1-WT,the NS1-S212P mutation inhibited the function of the H7N9 NS1 protein in antagonizing the host innate immune response,which activates the RIG-I-mediated immune response pathway;2)Compared with NS1-WT,the phosphorylation level of STAT1 was significantly increased after NS1-S212P mutation.NS1-S212P mutation provoked the host cell antiviral response and caused JAK-STAT signaling pathway to be activated and ultimately induced higher expression of several ISGs;3)NS1-S212P mutation resulted in reduced titers of influenza virus as compared with NS1-WT,indicating that NS1-S212P mutation might affect functioning of NS1 that alters the internal environment of host cell,thereby inhibiting the replication of influenza virus.The eukaryotic expression plasmid of H7N9 NS1 protein and its mutant(E70K,T143A,I178V,S212P)were transfected into 293T cells for 24 hours and the cells were treated with CHX(cycloheximide)for 32 hours.In order to verify the effect of mutation at position 1178 on NS1 protein stability,the expression of NS1 protein was detected by Western blotting.Subsequently,the stability of mutated NS1 protein after specific mutation was studied.293T cells were respectively treated with proteasome inhibitor(MG 132),lysosomal inhibitor(NH4CL,Chloroquine)together with CHX to detect the degradation pathway of NS1-I178V.The results are shown as follows:1)Compared with E70K,T143A and S212P mutations,the stability of H7N9 NS1 protein was decreased by 1178V mutation;2)The half-life of NS 1-1178V is about 16h,while the half-life of NS1-WT is more than 32h,further indicating that I178V mutation can reduce the protein stability of H7N9 NS1;3)The protein level of NS1-I178V was decreased after treatment with NH4Cl and Chloroquine.However,the degradation of NS 1-I178V protein was significantly inhibited by MG 132 treatment,indicating that 1178V mutation caused the NS 1 protein to undergo ubiquitin-proteasome pathway degradation.In summary,we identified that the S212 amino acid of H7N9 NS1 protein is a key site for NS1 in antagonizing the host antiviral immune response and the 1178 amino acid plays an important role in maintaining NS1 protein stability.These results provide novel insights into mechanism underlying the pathogenesis of H7N9 avian influenza virus and suggest a new idea for the drug development of anti-H7N9 avian influenza virus and the development of new vaccines.