| Influenza A viruses are enveloped viruses within the family Orthomyxoviridae, and they contain a single-stranded, negative sense, segmented RNA genome consisting of eight segments of viral RNA (vRNA). NS1 protein is encoded on a collinear mRNA derived from vRNA segment eight. It is not a structural component of the virion, but is expressed at very high levels in infected cells. The NS1 protein plays an important role in the regulation of viral pathogenicity and virulence, and also has impact on the immune response and apoptosis of host cells.The amino acid variations were analyzed between the NS1 protein in 2009 (2009NS1) and 1918(1918NS1) by DNAMAN software. Compared with 1918NS1, some variations were found in 2009NS1, such as PL domain deletion and 32 amino acid mutations.To further study NS1 influence on eukaryotic cell, 1918NS1 gene was got by gene synthesized method and 2009NS1 gene was cloned by RT-PCR method, 5 recombinant Eukaryotic expression vectors were constructed including TAPNS12009, HANS12009, HANS11918, GSTNS12009, GSTNS11918. The recombinant vector was transfected in A549 cells by Lipofectamine 2000 and the expression level of NS1 protein was detected by Western Blot method. The transfection conditions were optimized in order to get high level NS1 expression, including the different transfection reagent volume, plasmid quality dosage, transfection time course etc. And the result indicated the best transfection conditions were Lipofectamine 2000 9μL with plasmid 2μg. The NS1 protein expression leve was the highest, at the time after 96h of transfection.Transient transfection of 2009NS1, 1918NS1 eukaryotic expression vector in A549 cells, then the A549 cell morphology was observed by microscopy. It was found that shrunken, smaller sized, rounded, shrinkage and chromatin marginalization in A549 cell expressed NS1, which were typical phenomenon of cell apoptosis. AnnexinV staining was applied to detect the cells apoptosis, and the staining ratio of cells was higher in the experimental group than the blank and negative control group, meanwhile A549/1918NS1 staining ratio was higher than A549/2009NS1. The arrest in G2/M phase is detected in experimental groups by flow cytometry analysis, and the ratio of G2/M phase in A549/1918NS1 is greater than in A549/2009NS1. p53, p-p53-Ser15 and p21 were key proteins in cell cycle, its expression level were increased by western blot, which indicated NS1 upregulates these proteins, and the upregulation is greater in 1918NS1 than in 2009NS1. Above all these results suggest that NS1 protein play an important role in cell cycle arrest at G2/M phase and induced host cell apoptosis in A549 cell, and the possible mechanisms were by increasing the p53, p-p53-Ser15 and p21 protein expression level to regulate cell cycle and apoptosis. The influnce induced by 1918NS1 is greater than 2009NS1, maybe related on the PL domain deletion in 2009NS1.The fusion protein TAPNS1 was purified by TAP affinity purification technology, the interaction protein Nucleolin with NS1 was detected by Western blot. The interaction of NS1 and Nucleolin was verified by GST pull down experiment, and the interaction interface in the 1-82aa of NS1. Our results make a foundation for NS1 protein function research in cell cycle regulation and induction of apoptosis. |
PDF Full Text Request