PEDV拮抗IFN-Ⅰ和IFN-Ⅲ诱导的JAK/STAT信号通路的分子机制研究

Antiviral innate immune responses are induced by interferon (IFN)-activated JAK/STAT signaling pathway, while different strategies have been evolved to antagonize host antiviral responses. Porcine epidemic diarrhea virus (PEDV) is one of the most important pathogens in recent years, which caused economic loss in swine industry in our country. Our preliminary experiments showed that PEDV infection was not sensitive to IFN treatment in vitro and inhibited the transcription levels regulated by interferon-stimulated response element (ISRE), indicating that JAK/STAT signaling pathway is antagonized by PEDV infection. However, the antagonistic mechanism still remains unclear. Therefore, in this project, IFN-I and IFN-III-mediated JAK/STAT signaling pathways in IPEC-J2 and Vero E6 cells are investigated to analyze the receptors of IFN-I and IFN-III, expression and phosphorylation levels of key molecules in JAK/STAT signaling pathway, nuclear translocation and transcriptional regulation of transcription factor ISGF3, etc., to reveal the different ways of PEDV antagonizing JAK/STAT signaling pathway. In addition, we will also screen viral encoded/induced proteins with antagonistic activities followed by verification through protein interaction, reverse genetic manipulation techniques, etc., to elucidate the molecular mechanism of PEDV antagonizing JAK/STAT signaling pathway. This project will present a novel insight to PEDV vaccine development and drug treatment.

干扰素（IFN）通过激活JAK/STAT信号通路诱导宿主细胞抗病毒天然免疫应答，同时病毒进化形成不同策略拮抗宿主抗病毒反应。猪流行性腹泻病毒（PEDV）是近年来危害我国养猪业的重要病原之一，我们前期研究发现PEDV感染对IFN处理不敏感，并抑制ISRE元件调控转录，提示PEDV对JAK/STAT信号通路具有拮抗作用，但拮抗作用的分子机制尚不清楚。因此，本项目以IPEC-J2和Vero E6细胞IFN-Ⅰ和IFN-Ⅲ介导的JAK/STAT信号通路为研究对象，分析IFN-Ⅰ和IFN-Ⅲ受体、信号通路关键节点分子表达及磷酸化、转录因子ISGF3的核转运及转录调节等，揭示PEDV拮抗JAK/STAT信号通路的不同途径；筛选发挥拮抗作用的病毒蛋白/诱导蛋白，经蛋白互作、反向遗传操作技术等方法进行验证，阐明PEDV拮抗JAK/STAT信号通路的分子机制。本项目为PEDV疫苗研发和药物治疗提供新思路。

研究表明PEDV感染能够抑制宿主抗病毒应答，但PEDV对宿主天然免疫的拮抗作用尚不完全清楚。本项目发现PEDV感染可以明显下调内源性和外源FBXW7的表达，且FBXW7的下调作用依赖于病毒复制。为探索FBXW7对PEDV复制的影响，分别在Vero E6细胞和HEK293细胞中过表达和敲低FBXW7，发现过表达FBXW7能够显著抑制PEDV复制，敲低FBXW7能促进PEDV的复制，表明FBXW7为宿主细胞抗PEDV因子。进一步研究表明，FBXW7可以通过增加内源性视黄酸诱导基因-I蛋白（Retinoic acid-inducible gene I, RIG-I）和TBK1的表达来激活宿主干扰素信号通路。与siRNA NC转染的细胞相比，FBXW7 siRNA转染的靶细胞中的天然抗病毒分子（如IFNβ、ISG15、ISG54和ISG56）的mRNA水平均明显下调。为筛选与FBXW7相互作用的病毒蛋白，利用免疫沉淀质谱法（IP-MS）筛选发现PEDV的非结构蛋白nsp2可以直接靶向作用于FBXW7，进一步发现nsp2可以促进FBXW7发生K48的泛素化修饰，通过泛素蛋白酶体途径降解FBXW7。本研究揭示PEDV拮抗宿主天然免疫信号通路的一种新策略。