中文摘要：

目的探讨日本脑炎病毒抑制IFN-α介导的JAK-STAT信号转导通路过程中发挥关键作用的病毒特异蛋白,为阐明日本脑炎病毒抑制Ⅰ型IFN介导的JAK-STAT信号转导通路的作用机制奠定基础。方法分别构建日本脑炎病毒编码的7种非结构蛋白基因（NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5）的重组真核表达载体。采用间接免疫荧光法和Western blotting观察它们在细胞内的表达及定位情况。利用含萤火虫荧光素酶（Luc）报告基因的重组载体pISRE-Luc,观察表达病毒不同非结构蛋白的细胞内IFN-α介导的JAK-STAT信号转导通路的活化程度。构建融合表达红色荧光蛋白的STAT1重组真核表达载体pRed-STAT1,利用该重组载体在表达病毒非结构蛋白的细胞内观察融合蛋白Red-STAT1在IFN-α作用下的细胞内定位情况。同时,对表达病毒非结构蛋白的细胞内IFN-α介导的STAT1分子的磷酸化水平进行检测。结果日本脑炎病毒的7种非结构蛋白均可在哺乳动物细胞内正确表达,而且表达蛋白均位于细胞质中。在这7种非结构蛋白中,NS5可阻断STAT1分子的核转运及抑制STAT1分子的磷酸化水平,从而抑制IFN-α介导的细胞内JAK-STAT信号转导通路的活化。结论日本脑炎病毒的非结构蛋白NS5对Ⅰ型IFN系统的信号转导通路具有显著的抑制作用,可能是一种Ⅰ型IFN系统的拮抗蛋白。

英文摘要：

Objective To identify the Japanese encephalitis virus （JEV） non-structural proteins responsible for blocking IFN-α sig naling, and deeply understand the mechanism of the inhibition of IFN-α induced JAK-STAT pathway by JEV. Methods A series of ex pression plasmids of JEV non structural proteins were generated to individually express V5 tagged NS1, NS2A, NS2B, NS3, NS4A, NS4B and NSS, and the cellular distributions of these non-structural proteins were observed. The plSRE Luc was used to determine the ISRE activity and quantify the suppressive degree of JAK-STAT signaling in the cells expressing non：structural proteins of JEW. The expression plasmids pRed-STAT 1 were constructed, which encode the human STAT 1 fused in frame with a red fluorescent protein, by which the intraeellular status of JAK STAT signaling can be visualized. The cells were eo-transfected with plasmids expressing the indicated JEW non-structural proteins and pRed-STAT 1, and the status of Red-STAT 1 upon IF-α treatment was observed by immunofluorescence assay. The levels of IFN-α stimulated the STAT 1 phosphorylation in the cells expressing JEV non-structural proteins were analyzed by Western blotting. Results JEV non-structural proteins can correctly be expressed in mammalian cells and all the proteins located in the cytoplasm. JEV NSo clearly suppressed the ISRE activity, blocked nuclear translocation of Red STAT 1 fusion protein, and reduced the level of STAT 1 phosphorylation upon IFN-α treatment. Conclusion JEV NS5 can inhibit IFN-α induced JAK-STAT pathway and may be a potent IFN antagonist.