Effect Of Amino Acid Mutation Of Swine Influenza Virus NS1 Protein On Inhibition Of Interferon Production

Swine Influenza?SI?is a highly contagious mass of porcine respiratory diseases caused by Swine Influenza Virus?SIV?,which is distributed worldwide.Non-structural protein?NS1?is a protein of influenza A virus which plays a variety of functions during virus infection.The main role of NS1 is to inhibit the host immune response,especially the production of interferon?IFN?and the antiviral effect of IFN-induced proteins,such as dsRNA-dependent RNA-dependent protein kinase?PKR?and 2'5'-oligoadenylate synthetase?OAS?,in order to maintain viral infectivity.However,the key amino acid sites at which NS1 proteins play a role in different subtypes of SIV remain unclear.In this study,we took the swine influenza A/swine/Shanghai/3/2014?H1N1?isolate as template and used the site directed mutagenesis technique to carry out mutations in the important amino terminal sites of the Nuclear localization sequence?NLS?and the nuclear output sequence?Nuclear export signal,NES?of the NS1 protein.Mutant strains were rescued by reverse genetics technology.The distribution of mutant proteins and mutant viruses in cells,their effects on cytokine transcription and interferon production in mice were detected experimentally,which laid a foundation for further understanding the role of key amino acid sites of NS1.Five conservative key amino acids of NS1 protein NLS and NES were mutated to alanine,and five mutant NS1 plasmids(NS1_L36A,NS1_L141A,NS1_L146A,NS1_R38A38A and NS1_L144A)were constructed.Their localization in A549 cells was different.NS1_L36A,NS1_L141A141A and NS1_L146A146A were similar to wild-type NS1 protein,and the expression of protein was detected in both cytoplasm and nucleus.However,NS1_L144A144A protein mainly distributes in the cytoplasm and does not nucleate;a small amount of NS1_R38A38A enters the nucleus.These results suggest that the 144 and 38 amino acid sites in NS1 protein may affect the localization of NS1 protein.Further detection showed that the transcriptional levels of type I interferon??IFN-??and antiviral protein?OAS?were lower than those of wild NS1 protein entering cells,while the transcriptional levels of NS1 mutant protein NS1_R38A38A and NS1_L144A144A entered cells,and the transcriptional levels of intracellular type I interferon beta and antiviral protein?OAS?increased?P<0.05?.The reverse genetics platform of SIV was constructed and the virus titer of the mutant viruses rSIVNS1_R38A38A and rSIVNS1_L144A144A was decreased.Further,indirect immunofluorescence assay showed that NS1 protein was mainly localized in cytoplasm and nucleus in MDCK cells.IFN-?was found to be upregulated in A549 cells infected with rSIVNS1_R38A38A and rSIVNS1_L144A,whereas IFN-?was down-regulated in A549infected with rSIV and was inversely correlated with viral titer.Finally,experiments in mice showed that the viral titer of rSIV in mouse lungs was higher than that of rSIVNS1_R38A38A and rSIVNS1_L144A.Further detection of cytokine transcription levels in mouse lungs revealed that IFN-?and TNF-?were up-regulated in the lungs of mice after infection with rSIV,rSIVNS1_R38A38A and rSIVNS1_L144A144A viruses.In the lungs of mice infected with rSIV,IFN-?was down-regulated and negatively correlated with lung virus titer.In conclusion,this study confirmed that the 38th and 144th amino acid positions in the NS1 protein of the swine influenza A/swine/Shanghai/3/2014?H1N1?strain are key amino acid sites for the inhibition of interferon production by the viral NS1 protein.To lay the foundation for further study of the role of NS1 protein in inhibiting cellular innate immune response.