维甲酸诱导基因Ⅰ信号通路在西尼罗病毒感染中的作用研究

Role of retinoic acid-induced gene Ⅰ signal pathway during West Nile virus infection

目的探讨维甲酸诱导基因I(RIG-I)信号通路在西尼罗病毒感染过程中的作用。方法用西尼罗病毒Chin-01株感染A549细胞,分别通过RT-PCR和Western blotting检测病毒感染对RIG-I及干扰素β(IFN-β)启动子刺激因子-1(IPS-1)表达的影响,通过含CAT报告基因的重组质粒观察病毒感染对干扰素调控因子-3(IRF-3)和IFN-β启动子活性的影响,并用ELISA法检测IFN-β的表达水平,明确西尼罗病毒感染对RIG-I信号通路下游信号分子的激活作用。通过瞬时转染法在A549细胞中过表达RIG-I,观察其对西尼罗病毒感染的影响。结果西尼罗病毒感染能够上调RIG-I的表达,激活IPS-1、IRF-3等下游信号分子,诱导I型IFN的产生。同时,RIG-I的过表达能够抑制西尼罗病毒的增殖。结论RIG-I信号通路介导的固有免疫反应可能在西尼罗病毒感染过程中发挥重要作用。

Objective To explore the role of retinoic aNd-induced gene Ⅰ （RIG-Ⅰ） signal pathways during West Nile virus infection in vitra Methods Human lung epithelial cells A549 were infected with West Nile virus Chin-01 strain. RT-PCR and Western blotting were then performed to detect the expression of RIG-Ⅰ and IFN-β promoter stimulator （IPS-1） genes 24h after infection. And two CAT reporter plasmids （PRDⅢ-D3-CAT and （-110-IFNβ）-CAT were then used to transfect the infected A549 cells, respectively, and the activity of interferon regulatory factor 3 （IRF-3） and IFN-β promoter were determined by CAT ELISA assay. The expression level of IFN-β in the culture supernatant was further measured by a human ELISA kit. In another set of experiment, A549 cells were infected with West Nile virus at different MOI, then the antiviral effects of RIG-Ⅰ expression were observed. Results Both RT-PCR and Western blotting results suggested that RIG-Ⅰ were up-regulated by West Nile virus, and the downstream molecules of RIG-Ⅰ signal pathway, IPS-1, IRF-3 and IFN- β, were all activated in West Nile virus infection. What is more, over-expression of RIG-Ⅰ gene in A549 cells was found to reduce the pro duction of newly produced West Nile virus. Conclusion RIG-Ⅰ signal pathways were induced by West Nile virus infection and might play an essential role during virus recognition and antiviral innate immunity response.