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目的探讨柯萨奇病毒A组16型对RIG-Ⅰ介导的信号通路的影响。方法将带有IFN-β启动子的萤火虫荧光素酶报告质粒pIFN-β-luc及内对照报告载体pRL-CMV转染细胞,之后用CA16感染细胞,24 h后检测报告基因活性。将CA16感染细胞,在感染6 h、12 h、24 h以及48 h后用Real-time PCR方法测定细胞中IFN-β的mRNA的表达水平。将pIFN-β-luc和pRL-CMV以及表达RIG-I的质粒和表达CA16的3C蛋白质粒共转染细胞,24 h后检测报告基因活性。将带有绿色荧光蛋白GFP的IRF3质粒和表达RIG-I基因N端质粒以及表达CA16的3C蛋白共转染细胞,24 h后用荧光显微镜观察。将表达RIG-I的质粒和表达CA16的3C蛋白质粒共转染细胞,24 h后用免疫共沉淀方法验证蛋白质间相互作用。结果 CA16感染细胞不能引起IFN-β表达的上调,CA16的3C蛋白抑制了RIG-I诱导的IFN-β的启动子活性以及IRF3的核迁移,CA16的3C蛋白与RIG-Ⅰ存在相互作用。结论 CA16的3C蛋白通过与RIG-Ⅰ相互作用从而抑制RIG-Ⅰ介导的IFN-β的诱导。
Objective To investigate the effect of coxsackievirus A group 16 on RIG-Ⅰ-mediated signaling pathway. Methods The firefly luciferase reporter plasmid pIFN-β-luc with IFN-β promoter and pRL-CMV vector was transfected into the cells. The cells were infected with CA16 and the activity of reporter gene was detected after 24 hours. The cells were infected with CA16, and the mRNA expression of IFN-β in the cells was detected by Real-time PCR at 6 h, 12 h, 24 h and 48 h after infection. The cells were cotransfected with pIFN-β-luc and pRL-CMV as well as the RIG-I expressing plasmid and the 3C protein expressing CA16, and the reporter activity was detected 24 h later. The IRF3 plasmid with green fluorescent protein (GFP) and the N-terminal plasmid expressing RIG-I gene and the 3C protein expressing CA16 were co-transfected into the cells and observed by fluorescence microscopy after 24 hours. Cells were co-transfected with the plasmid expressing RIG-I and the 3C protein expressing CA16, and the protein interaction was verified by co-immunoprecipitation 24 hours later. Results The CA16-infected cells did not induce the up-regulation of IFN-β expression. The 3C protein of CA16 inhibited the promoter activity of IFN-β induced by RIG-I and the nuclear translocation of IRF3. The 3C protein of CA16 interacted with RIG-Ⅰ. Conclusion The 3C protein of CA16 can inhibit RIG-Ⅰ-mediated induction of IFN-β by interacting with RIG-Ⅰ.