柯萨奇病毒A组16型的3C蛋白抑制RIG-Ⅰ介导的IFN-β的诱导

Inhibitory effects of coxsackievirus A16 3C protein on RIG-Ⅰ-mediated induction of IFN-β

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。

The retinoic acid-inducible gene Ⅰ（RIG-Ⅰ） has been identified as a cellular sensor of RNA virus infection resulting in IFN-β induction.However,many viruses are known to encode viral products that inhibit IFN-β production.Coxsackievirus A16（CA16） is a major causative agent of hand,foot,and mouth disease,while we aim to investigate the effect of RIG-Ⅰ signal transduction by CA16.Reporter gene assay and Real-time PCR analysis were used to detect the effect of CA16 infection on IFN-β production in cells.Furthermore,cell transfection,reporter gene assay and IRF3 nuclear translocation assay were used to detect the effect of CA16 3C protein on RIG-I-mediated induction of IFN-β promoter activation and IRF3 nuclear translocation.Interaction of RIG-Ⅰ and CA16 3C protein was detected by immunoprecipitation.The results showed that CA16 infection does not induce IFN-β production,while the 3C protein of CA16 inhibits RIG-I mediated induction of IFN-β promoter activation and IRF3 nuclear translocation.All the results indicated that the 3C protein of CA16 could inhibit RIG-Ⅰ-mediated activation of IRF3 and induction of IFN-β by interacting with RIG-Ⅰ.