MERS冠状病毒蛋白酶调节复制酶复合体功能及其在抗病毒天然免疫中的作用机制

The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in human, and represents the second example of a highly pathogenic coronavirus followed SARS-CoV. MERS coronavirus infection in human is characterized by an aberrant immune response, as MERS-CoV fails to elicit strong type I interferon or pro-inflammatory innate immune responses, suggesting that MERS-CoV has developed some strategies to evade and subvert the host antiviral innate immune response. However, the mechanisms used by MERS-CoV to evade and subvert the host antiviral innate immune response are not fully understood. Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and interferon antagonist, but the function and their roles in viral infection and antiviral innate immunity of the MERS-CoV protease and replicase complex (RC) were poorly understood. In this project, we hypothesis a new mechanism that human MERS coronavirus protease and replicase complex modulate infection and antiviral innate immunity through targeting the specific host proteins and signaling pathways. In this study, we will propose: 1) to determine the functions of MERS-CoV papain-like protease (PLpro) and 3CLpro and their roles in production and regulation of replicase complex (RC). 2) to determine the regulation mechanism of RC in viral RNA replication and transcription. 3) to determine the relationships of RC with double membrane vesicle (DMV) and exosome and its roles in regulations of viral infections and innate antiviral immunity, and 4) to determine the regulation mechanisms of antiviral innate immunity through targeting cellullar signaling protein complex by MERS protease and RC. This project will provide new information on infection and antiviral immunity regulations of the new emerging MERS-CoV and will identify potential targets for antiviral interventions for human coronavirus infection.

MERS冠状病毒是2012年首次出现的又一种人类高致病性新型冠状病毒,致死率远超SARS冠状病毒. 目前对MERS-CoV感染与免疫逃避机制还不清楚.申请者在国家自然科学基金持续资助下,对SARS等新发冠状病毒蛋白酶生物学功能及其调节宿主抗病毒天然免疫机制进行了深入系统研究。本课题以前期研究为基础，针对MERS-CoV感染与免疫抑制(逃避)这一科学问题，提出“复制酶复合体(RC)通过特定宿主蛋白和细胞信号通路调节抗病毒天然免疫”科学假设。课题拟以MERS-CoV为对象，应用分子生物学和免疫学技术研究:MERS-CoV蛋白酶生物学功能及其参与RC组装和酶活性调节机制;RC 参与双层膜囊泡(DMV)形成及其与外泌体功能联系;蛋白酶和RC调节抗病毒天然免疫及其机制. 课题对研究MERS-CoV蛋白酶和RC功能,揭示人类冠状病毒感染与免疫调节新机制具有重要理论意义。

项目通过四年实施，系统研究MERS-CoV 两种重要蛋白酶PLpro和3CLpro的生物学功能及作用机制，完成了研究内容，取得了系列创新发现：MERS-CoV编码的PLpro和3CLpro具有催化生成nsp调节病毒复制酶复合体RC形成，负调控宿主抗病毒天然免疫，诱导宿主细胞自噬的功能：（1）建立了基于生物传感原理的蛋白酶活性测定模型，利用该模型系统研究MERS PLpro和3CLpro的催化特性，发现了蛋白酶识别底物保守序列的特性，以及不同冠状病毒蛋白酶的活性差异。（2）利用该模型初步筛选到MERS 3CLpro候选抑制剂Cinaserin。（3）发现MERS 3CLpro 能够负调控宿主干扰素抗病毒天然免疫，通过RIG-I通路抑制I型干扰素表达，其干扰素拮抗活性依赖于蛋白酶活性位点（C3395和H3288）。（4）发现MERS 3CLpro蛋白酶具有去泛素化（DUB）活性，能够去除细胞内蛋白质的Ub、Ub-K48和Ub-K63等形式泛素化以及类泛素分子ISG15和SUMO的修饰，其DUB活性关键位点与蛋白酶活性位点（C3395和H3288）相同；MERS 3CLpro能够去除IFNβ表达通路重要调节蛋白RIG-I､TBK1､IRF3的泛素化修饰，这可能是其负调控抗病毒天然免疫的机制之一。（5）发现MERS 3CLpro蛋白酶能够诱导宿主细胞发生不完全自噬，激活自噬体形成过程而对抑制自噬溶酶体的成熟过程；其自噬诱导活性具有时间依赖性且不依赖酶活性位点。总之，发现MERS-CoV的PLpro和3CLpro蛋白酶都是病毒编码的天然免疫拮抗剂，能够负调控宿主干扰素抗病毒免疫，机制可能在于蛋白酶与IFNβ表达通路重要调节蛋白RIG-I、MAVS、STING、TBK1、IRF3 等发生相互作用，去除后者的泛素化修饰，抑制其信号转导和I型IFN生成，并诱导宿主细胞自噬。

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