病毒响应蛋白OTUD3调控MAVS抗病毒免疫信号的研究

The viral infectious diseases emerge to be a great threat to the public health. It is of great significance to develop clinical prevention tools by screening out essential regulators of antiviral immunity and systematically reveal their working mechanisms. Although multiple deubiquitinases (DUBs) were shown to be involved in innate antiviral response, weather DUB activity could be modulated by viral infection is unknown. This project aimed to screen out crucial DUBs that are regulated by viral infection and we will also examine how their altered DUB activity could affect viral amplification. We have established assays by using specific chemical probes to capture the active DUBs in vivo. This allows us to compare DUB activity at the endogenous level. Combined with proteomic quantitative analysis experiments, our preliminary result showed that the OTUD3 activity could be strikingly inhibited upon viral infection. Based on these observations, we plan to address questions below: 1) Why the OTUD3 activity needs to be suppressed? 2) How could OTUD3 response to viral infection, weather this is due to post-translational modifications; 3) Which kind of protein modification is required for above-mentioned process; 4) Weather OTUD3 also in turn regulates RLRs-signaling pathway. We also aim to further find how the activity of OTUD3 is regulated by intracellular metabolic activity and whether DUB activity could be inhibitor by small chemicals. Upon successful conclusion of this work, we will have provided not only new regulators for innate immunity, but also proposed new medical strategies to enhance antiviral immunity.

病毒性传染病是现代人类公共健康的主要威胁。如何系统快速地筛选出潜在的抗病毒免疫关键因子并揭示其作用机理对于预防和治疗病毒性疾病具有重要意义。关于DUB在抗病毒免疫中的功能和调控虽有报道，但哪些内源的DUB在先天免疫激活时发挥活性或受到调控还不是很清楚。申请人准备利用体内鉴定活化型DUB的策略结合蛋白质质谱技术筛选整个DUB家族，从而获得病毒感染后特异活化或失活的DUB。筛选结果显示，OTUD3活性被极大抑制。本项目计划进一步探究OTUD3响应病毒感染变化的分子机制和其对RNA病毒-MAVS信号通路的调控作用。回答在病毒感染的过程中，OTUD3的活性是如何被蛋白修饰所调控的？是如何调控RLRs-MAVS抗病毒信号的？此外，OTUD3的活性是否受到细胞内代谢活性的调控？本项目旨在发现新的抗病毒调控因子，揭示DUB调控抗病毒免疫新的机制，为临床治疗和药物筛选提供给理论基础。

病毒性传染病是现代人类公共健康的主要威胁，探明抗病毒免疫的分子机制是预防和治疗病毒性疾病的关键。如何系统快速地筛选出潜在的抗病毒免疫关键因子并研究其作用机理对于临床防治和药物研发具有重要的意义。本课题建立了两种泛素Ub化学探针捕获体内活性去泛素化酶(DUBs)的技术，并结合报告基因功能实验，系统性地探究了病毒感染前后整个去泛素化酶家族的酶活变化，揭示了响应RNA病毒感染的活性DUB的谱图变化，发现OTUD3的活性在病毒感染后被严重抑制。Otud3敲除的小鼠表现为增强的抗病毒先天免疫反应，机制上，OTUD3切割MAVS的K63连接的多聚泛素化，抑制其聚集和对下游信号蛋白的招募，进而抑制抗病毒先天免疫应答。同时OTUD3的活性受到乙酰化的严格控制，其催化活性依赖于129位赖氨酸的乙酰化。当宿主细胞未被病毒感染时，OTUD3被乙酰化并保持高活性，抑制先天免疫；而当病毒感染时，去乙酰化酶SIRT1被招募到细胞质与OTUD3结合，去乙酰化OTUD3使其迅速失活，从而及时激活抗病毒免疫。这些发现证明OTUD3是MAVS的一个负调控因子，并揭示了一个全新的通过乙酰化调控DUB活性的机制，通过该机制严格控制OTUD3的催化活性，以确保宿主及时激活抗病毒防御。