去泛素化酶USP4通过RLR信号通路调控肠道病毒71型感染的机制和功能研究

RIG-I like receptor（RLR）is the key element that senses viral RNA in cytoplasma, and its members include RIG-I, MDA5 and LGP-2. It has been reported that deubiquitinase USP4 can specifically remove activated RIG-I ubiquitin chains and inhibit virus replication in vesicular stomatitis virus infection. Whereas, in TNF-α signal, USP4 can deubiquitinate TRAF2/6. But little is known about the function of USP4 and its relationship with enterovirus 71 (EV71) infection. Our preliminary studies show that EV71 infection could activate MDA5 signaling pathway, and USP4 inhibits the production of EV71-induced IFN-β in RNAi screening. In the present study, we will explore the relationship between USP4 and EV71 infection through the molecular，cellular，and animal levels. To observe the effect of USP4 on the release of interferon and inflammation-related factors, and further study the regulatory mechanism of USP4 deubiquitination on MDA5 and TRAF2/6 during EV71 infection，which will provide new potential targets for the treatment of EV71 infection. Meanwhile, this study will also give an experimental basis for assessing the curative effect and prognosis by analyzing the serum levels of USP4 mRNA and protein in patients with EV71 infection.

RIG-I样受体（RLR）是识别胞浆中病毒RNA的主要元件，其成员包括RIG-I、MDA5和LGP-2。研究表明，去泛素化酶USP4可对水泡性口炎病毒感染激活的RIG-I去泛素化修饰并抑制病毒复制，而在TNF-α信号中USP4能去泛素化TRAF2/6。但USP4的功能及其与肠道病毒71型(EV71)感染的关系尚未明确。. 我们前期工作发现，EV71感染可激活MDA5信号通路，RNAi筛选也显示USP4能抑制EV71诱导的IFN-β产生。本项目拟从分子、细胞、动物等水平探讨USP4与EV71感染的关系，观察USP4对EV71感染下干扰素和炎症相关因子产生的影响，深入研究USP4对EV71感染活化的MDA5和TRAF2/6去泛素化调节机制，为治疗EV71感染提供潜在的新靶标。同时，通过分析EV71感染患者血清中USP4 mRNA及蛋白水平，为评估疗效及预后提供实验依据。

背景：EV71感染所致手足口病，多发生于5岁以下儿童，少数患儿可引起心肌炎、肺水肿、无菌性脑膜脑炎等并发症，重症病例常导致死亡。针对EV71感染的防治主要有疫苗、抗病毒化学合成药物及外源性细胞因子等，但临床应用效果仍不理想。阐明去泛素化酶USP4对EV71感染的固有免疫调控机制，为治疗EV71感染提供行的策略和途径。.主要研究内容：应用PCR芯片技术检测EV71感染人横纹肌肉瘤（RD）细胞不同时间点88种去泛素化酶基因差异表达，免疫印迹法分析RLR信号通路关键分子磷酸化水平；RNA干扰、免疫共沉淀等技术分析去泛素化酶USP4通过剪切K-48或K-63多聚泛素化链调节NF-κB信号。荧光定量PCR检测40例患儿血清中USP4/mRNA水平。并采用CRISPR/Cas9技术敲除小鼠USP4基因，PCR扩增并测序鉴定纯合子小鼠,构建USP4-/-模型。.重要结果：EV71在体内外感染宿主细胞可致去泛素化酶USP4表达下调，而USP4过度表达可显著抑制EV71的增殖。EV71中的2A蛋白酶能切割USP4蛋白。同时发现EV71感染降低了RLR信号通路并增强了TRAF6的降解。去泛素化酶USP4靶向作用于TRAF6蛋白，并剪切其K48多聚泛素化而上调NF-κB信号。EV71感染患儿血清USP4/mRNA水平明显降低，与对照组相比，差异有统计意义（P<0.05）。USP4-/-小鼠繁育未获成功,敲除的USP4基因是否影响小鼠生育须进一步加以研究。.关键数据及其科学意义：去泛素化酶USP4通过去除TRAF6蛋白K48多聚泛素化而正性调控NF-κB信号,从而证实USP4参与细胞中抗EV71感染天然免疫反应，利于EV71逃避宿主固有免疫应答。建立的荧光定量PCR法检测EV71患者血清中去泛素化酶USP4 mRNA，可望为快速诊断EV71感染提供依据。