The Research On Synergistic Effect Of The Key Interaction Sites Between NS1 Protein Of Avian Influenza(H5N1)and NOLC1 Of Host Protein

Non-structural protein 1(NS1)is an important virulence factor encoded by influenza A virus(IAV).NS1 can interact with a variety of host cell proteins to interfere with the host innate immune response and to promote effective viral replication and regulate host cell apoptosis.Our preliminary work,the T7-phage display technology has been used to screen novel cellular factors that interact with NS1,which is human nucleolar and coiled-body phosphoprotein 1(NOLC1),On this basis,the gene truncation techniques,His-pull down,PCR site-directed mutagenesis were used to determine the amino acids 104-200 minimum area of NS1 effects domain(ED)terminus is accurate area of the NS1-NOLC1 protein interaction,further determine the NS1(ED)area 120-D and 195-R acid are key sites involved in NS1-NOLC1 protein interaction.To research on 120-D and 195-R synergistic effect of the key interaction sites between NS1 protein and host protein NOLC1,PCR site-directed mutagenesis was used to mutation double point 120 D and 195 R,and GFP-NS1-ED(D120A / R195A)double mutation eukaryotic expression vector also was construct and transfected into COS-7 cells obtained proteins.His-pull down techniques was used to determine NS1 variety mutant protein interaction with NOLC1,the results show,the interaction of NS1 double point mutation protein and NOLC1 was weakened,which reduced by about 83% compared to wild type NS1,indicating that 120 D and 195 R amino acid sites of NS1(ED)have synergistic effect on NS1-NOLC1 interact.On this basis,in order to further study the biological function of the interaction of NS1-NOLC1,full-length expression plasmid GFP-NS1,double point mutant plasmids GFP-NS1-ED(D120A/R195A),expression plasmid GFP-NOLC1 and interference expression plasmid shRNA-NOLC1 were respectively transfected in A549 cells,and Hochest33342 staining and AnnxinV-APC/PI double staining flow cytometry were used to qualitatively and quantitatively detect A549 cell apoptosis,the results show Apoptosis rate of A549 cells transfected with the full-length NS1 plasmid was 65.5%,while two-point mutation transfected NS1(120D/195R)of A549 cells was 29.7%,it can be seen from the experimental results,and compared with the full-length NS1 protein NS1(120D/195R)double point mutation protein,A549 cells apoptosis rate decreased;The overexpression and inhibits expression NOLC1,which the apoptosis rate is 56.4% and 44.0%,respectively,indicating that the protein expression of NOLC1 in A549 cells is closely related to the cells apoptotic,which is too high or too low will influence apoptosis,NS1-NOLC1 interaction is likely to be one of critical path of NS1 protein induces apoptosis in host cells,however,it need further study.NS1(ED)region 120-D and 195-R synergies in the interaction of NS1 and NOLC1 was identified,the biological function of NS1-NOLC1 interaction was explored,which provides theoretical basis for further study of the interaction between the two proteins and provided a new way of thinking for analysizing pathogenic of avian influenza virus.