Subcellular Localization of Nonstructural Proteins of Duck Tembusu Virus and Their Role in IFN-β Signaling Pathway
[Objective]This study was aimed to explore subcellular localization of nonstructural proteins of Duck Tembusu virus(DTMUV)and their role in the β-interferon(IFN-β)signaling pathway.[Method]7 nonstructural protein genes(NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5)of DTMUV were amplified by RT-PCR,cloned into eukaryotic expression vector pCAGGS-HA,constructed recombinant eukaryotic expression plasmid,and transfected into HEK-293T cells,respectively.The proteins expression was detected by Western blotting.The subcellular localization of nonstructural proteins was detected by indirect immunofluorescence assay,and the effect of DTMUV nonstructural proteins on the activity of IFN-β promoter from ducks was studied by dual luciferase reporting assay.[Result]The experiment successfully constructed 7 eukaryotic expression plasmids of DTMUV with HA tags for nonstructural proteins.Western blotting results showed that the eukaryotic expression of nonstructural proteins was normal,and the molecular weights of NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5 proteins were 38,25,14.4,68,13.9,28 and 100 ku,respectively.The results of indirect immunofluorescence assay showed that the expression patterns of the 7 nonstructural proteins were different in the cell,mainly located in the cytoplasm.The results of dual luciferase reporting assay showed that compared with control group,the activity of IFN-β promoter from ducks was extremely significantly decreased after overexpression of NS2B and NS4B proteins(P＜0.01).[Conclusion]In this study,the eukaryotic expression plasmid of 7 nonstructural proteins of DTMUV was successfully constructed,and the nonstructural proteins were mainly expressed in cytoplasm,among which NS2B and NS4B proteins had the function of opposing IFN-β activity.The results provided a theoretical basis for the study of immune escape of DTMUV,and provided experimental materials for further exploring the mechanism of action of DTMUV in host natural immune signaling pathway.
Duck Tembusu virusnonstructural proteinsubcellular locationIFN-β signal pathway
万方数据
维普
