Effect Of Site-directed Mutageneses Of Amino Acids In The Effector Domain Of NS1 Protein Of H1N1 Subtype Influenza Virus

According to the statistics of the World Health Organization,there are hundreds of millions of people infected with influenza virus every year in the world.In addition to the seasonal influenza virus at ordinary times,there are also occasional outbreaks of pandemic influenza due to new variant influenza strains.Two of the four global influenza pandemics in human history?H1N1 in 1918 and H1N1 in 2009?are caused by H1N1 virus,which has the characteristics of wide host range and large epidemic area and seriously endangers the livestock and poultry industry and human health in China.Therefore,it is of great epidemiological and public health significance to study the molecular mechanism of H1N1 virus pathogenicity.NS1 protein of IAV plays an important role in regulating gene expression,inhibiting innate immune response of host and influencing pathogenicity of virus.NS1 protein effector region inhibits the production of mRNA,especially IFN-?mRNA,by inhibiting the processing of 3'end of precursor mRNA in cells.The amino acid sequence of 10,240 H1N1 viruses in GenBank showed that there were significant differences in the 108th,123th,125th and 189th amino acid sites of NS1.The dominant amino acids of NS1 protein of 1,554 strains were 108K,123I,125D and 189D,and those of 8686 strains were 108R,123V,125E and 189G.In this study,influenza strain A/Puerto Rico/8/1934?H1N1?was used as the female parent to carry out amino acid co mutation?named PDM?at these four sites,and then carry out amino acid unit point mutation at these four sites,namely R108K,V123I,E125D and G189D,respectively.Then,the non-mutated NS1 recombinant expression plasmid pCAGGS-NS1_wtt and the mutated NS1 recombinant expression plasmid pCAGGS-NS1_mutut were constructed,including pCAGGS-NS1_R108K,pCAGGS-NS1 _V123I,pCAGGS-NS1_E125D,pCAGGS-NS1_G189D,pCAGGS-NS1_pdm.The wild-type non-mutated virus ReNS1_wtt and five site variant viruses ReNS1_R108K,ReNS1_V123I,ReNS1_E125D,ReNS1_G189D,ReNS1_pdmdm were rescued by reverse genetic technology.The double luciferase experiment showed that virus infection on IFN-?and its induced gene expression experiment explored the effect of amino acid variation on IFN-?transcription activity.The experimental results showed that compared with NS1_wtt group,the mRNA transcription level of IFN-?in NS1_R108K108K group and NS1_V123I123I group was significantly inhibited;the mRNA transcription level of IFN-?in NS1_G189D189D group and NS1_pdmdm group was inhibited;the mRNA transcription level of NS1_E125D125D group was inhibited,It was slightly down-regulated,and the inhibitory effect was increased.Flow cytometry was used to investigate the effect of plasmid transfection and virus infection on apoptosis.The results showed that NS1_wtt group had the strongest ability of inducing apoptosis in early stage,and the apoptotic cells were significantly more than other groups.The number of apoptotic cells in late stage increased with the extension of infection time.In the late stage of infection,the ability of inducing apoptosis in NS1_wtt group and NS1_E125D125D group was higher than other groups.The results showed that the latent period of pathogenicity of NS1_wtt and NS1_E125D125D groups was shorter,and the pathogenicity of the virus was stronger than that of other groups.The results showed that the 125th amino acid variation of NS1 protein effect region is an important amino acid site that affects virus antiviral response,virus replication and proliferation,cell apoptosis and virus pathogenicity.In this study,the effect of amino acid site variation of NS1 protein effect region on the virus was discussed preliminarily,which deepened people's understanding of the function of NS1 protein of influenza virus,and laid a theoretical foundation for better prevention and control of influenza transmission and avoidance of human and animal infection.