不同亚型流感病毒NS1蛋白的细胞定位差异分析

Comparison of cellular localization of NS1 from different subtypes of influenza A virus

摘要:

NS1蛋白是流感病毒编码的一种小分子多功能蛋白，可在病毒的复制过程中抑制宿主细胞的抗病毒免疫应答。为研究不同亚型流感病毒的NS1蛋白在细胞内的定位差异，分别用H1N1亚型WSN、PR8和CA04毒株，H9N2亚型SD毒株及H7N9亚型AH01毒株感染A549、MDCK细胞系以及构建的可表达不同亚型流感病毒NS1蛋白的pCMV-Myc-NS1质粒转染293T细胞，用激光共聚焦显微镜观察发现不同亚型流感病毒在不同细胞系和时间点的定位差异，感染后24 h时WSN和PR8毒株的NS1主要定位于细胞质中，而CA04和SD毒株主要定位于细胞核内。另外，观察过表达的WSN、SD和AH01毒株NS1的细胞定位，转染后24 h时WSN毒株NS1定位于细胞质中，而SD和AH01毒株主要定位于细胞核中。经氨基酸序列比对，对WSN毒株NS1蛋白进行关键氨基酸点突变，结果显示单一位点的改变未导致NS1蛋白细胞定位的改变，其细胞定位的差异不是由单一位点决定的。综上所述，分析不同亚型中的NS1的定位差异，这对进一步了解NS1 蛋白同宿主细胞不同区域的蛋白的相互作用、流感病毒的调节机制以及病毒感染细胞中天然免疫反应具有一定的指导意义。

Abstract:

The non-structural (NS1) protein is a multifunctional molecular protein encoded by influenza A virus genome. NS1 plays an important role in inhibition of host immune responses. In order to assess the cellular localization of NS1 in different influenza A virus subtypes, we performed the immunofluorescence assay to observe the cellular location of NS1 after infection with influenza A virus WSN (H1N1), PR8 (H1N1), CA04 (H1N1), SD (H9N2) and AH01 (H7N9) in A549 cells and MDCK cells respectively. According to the results, NS1-WSN and NS1-PR8 accumulated mainly in cytoplasm at 24 h post infection, while the NS1-CA04 and NS1-SD appeared major in the nucleus. We also observed localization of NS1 by transfected 293T cells with plasmids which encoding the full-length NS1 from WSN, SD and AH01. The key sites which might determine the different cellular localization of NS1 were chosen by sequence alignment, and seven residues which were different between WSN, PR8 and CA04, SD and AH01 were finally focused. However, we found that single mutation of these residues could not alter the localization of NS1. The data indicated that the difference of location might not be caused by substitution of a single site, which contributes to our understanding of the diverse regulation of host factors during different subtypes of influenza virus infection.