The Preliminary Study On The Function Of Avian Influenza A H7N9 Virus NS1

Since early 2013,zoonotic transmission of a novel avian-origin H7N9 influenza A virus has led to hundreds of human infections and deaths in China,which poses a big threat to human life.The influence of novel H7N9 virus NS1 on viral replication and host innate immunity were studied in this paper.By four amino acids mutation of NS1 protein,we try to find the key amino acids sites of NS1.Our study provides a new strategy and theoretical basis to develop antiviral medicine to antagonize the novel H7N9 virus infection.The NS1 of A/Shanghai/2013(H7N9)strain was cloned into the eukaryotic expression vector pNL-CMV-GFP.Then we transfected the constructed pNL-NS1 into 293T cells,and infected the cells with A/WSN/33(HIN1)virus to study the H7N9 NS1 influence on the viral replication and host innate immunity during the influenza a virus infection.We mutated the amino acids at 70?143?178?212 sites of the A/Shanghai/2013(H7N9)strain NS1 protein,and constructed the recombinant plasmids:pNL-NS1-E70K?pNL-NS1-T143A?pNL-NS1-I178V and pNL-NS1-S212P.Then pNL-NS1-WT and pNL-NS1-I178V were transfected into 293T cells.After treated with CHX for 0h?6h?12h?18h?24h?36h,we detected the half-lives of NS1-WT and NS1-I178V.The pNL-NS1-E70K?pNL-NS1-T143A?pNL-NS1-S212P were transfected into 293T cells,and infected with A/WSN/33(H1N1)virus to detected the effect of NS1-E70K?NS1-T143A?NS1-S212P on the host antiviral response.The results showed that the novel H7N9 NS1 could significantly increase the viral titers and viral protein synthesis in the influenza-infected cells.In addition,H7N9 NS1 inhibited the activities of RIG-I and its downstream factor IPS-1 and transcription factor NF-?B,limited the production of IFN and ISGs.To conclude,the novel H7N9 virus NS1 protein inhibits the host innate immunity by blocking RIG-I-depended signal pathway.4 amino acids mutation vectors of A/Shanghai/2013(H7N9)strain NS1 protein were constructed successfully:pNL-NS1-E70K?pNL-NS1-T143A?pNL-NS1-I178V and pNL-NS1-S212P.After treated with CHX for 18h,the protein level of NS1-I178V decreased 75%,but what of NS1-WT only reduced 36%.The result indicated that the I178V substitution remarkably reduced the stability of H7N9 NS1;Additionally,compared with the wild NS1,the NS1-S212P caused a significant increasing of the IFN and ISGs,which indicated that the S212P mutation weakened the inhibition of host antiviral response by H7N9 NS1.Whereas NS1-E70K and NS1-T143A had no significant influence on the functions of NS1.As a conclusion,the novel avian-origin H7N9 influenza A virus NS1 increases the viral titers and viral proteins synthesis in the influenza-infected cells,and inhibits the host innate immunity by blocking RIG-I-depended signal pathway.The stability of H7N9 NS1 requires the Iso-178,and Ser-212 plays a key role in its host innate immunity inhibition.Otherwise,the 70 and 143 amino acids substitutions may have no functional influence on H7N9 NS1.