Effect Of Key Amino Acids Of Swine In Influenza Virus NS1Protein On Nuclear Localization And Cytokine Production

Swine influenza(SI) is an economically important respiratory disease of swine resulting from infection with influenza A virus.Non-structural1(NSl) is a key regulatory protein and virulence factor in influenza A virus, which developed multiple ways to antagonize the host immune defense.Some studies have found that specific amino acids of NS1might be important for virus to escape host immune defense.lt has been reported that amino acid42can significantly influence the expression of IFN-P and amino acid92also play a key role in the virus virulence.In the study, NS1gene of A/swine/Shanghai/1/2007was amplified by RT-PCR and the.The amino acids of42S and92D were mutated into42P and92E respectively by SOEing PCR. The positive recombinant plasmids were identified by endonuclease digestion and sequencing. Then the NS1and mutated NS1genes were cloned into an eukaryotic expression vector pCAGGS. The recombinants plasmids were named pCAGGS-NS1pCAGGS-NS1S42P pCAGGS-NS1D92E and pCAGGS-NS1S42P/D92E. The purified recombinant plasmids were transfected into293T and A549cells respectively. SDS-PAGE and Western blot analysis indicated that four recombinant proteins were expressed successfully. The recombinant proteins were localized mainly in the nuclear and some was observed in cytoplasma using a fluorescence microscope. In addition, the interaction between the NS1protein and NP protein,Semiquantitative detection methods for IFN-α/β mRNA and TNF-a mRNA of human cell were established using total RNA of293T cells stimulated with Poly(I:C). After transfection of plasmids, IFN-α/βmRNA and TNF-amRNA of cells were detected using semiquantitative PCR. ELISA was used for IFN-α/β and TNF-a detection. The results showed that NS1S42P could significantly improve the IFN-p mRNA (P<0.01)and IFN-a (P<0.05), mRNA levels, but had no obvious influence on TNF-a mRNA level(P>0.05). The plasmids pCAGGS-NS1D92E and pCAGGS-NS1S42P/D92E transfection did not change the IFN-α/βand TNF-amRNA levels in the cells compared with the transfected cells with pCAGGS-NS1(P>0.05).The ELISA results showed that S42P could significantly improve the expression of IFN-β(P<0.01)and IFN-a(P<0.05), but had no obvious influence on TNF-a(P>0.05). Furthermore IFN-α/β and TNF-aproduced in the cells transfected with pCAGGS-NS1D92E or pCAGGS-NS1S42P/D92E had almost the same levels with those of the cells transfected with pCAGGS-NS1(P>0.05).There are two different kinds of NS1protein in swine influenza virus,219aa and230aa in length, respectively.In order to know if there are difference on cytokine production between these two kinds of NS1proteins, we selected A/swine/Zhejiang/2/2007(219aa) and A/swine/Shanghai/1/2007(230aa) to infect293T cells and detected the cytokine production. IFN-α/β and TNF-awere examined by ELISA24hours post infection. The results showed that both SIV could significantly improve the expression of IFN-α/β and TNF-a.There was no difference between these two groups. Furthermore BALB/c mice were infected with the A/swine/Zhejiang/2/2007and A/swine/Shanghai/1/2007respectively, and at48hours after infection, T cells of spleen48hours later infection were used for detection of CD8+and CD4+of T lymphocytes by flow cytometry. Both SFV strains could significantly improve the amouts of CD8+and CD4+of T lymphocytes and CD4+/CD8+ratio. But there was no difference between two gruops.