SARS冠状病毒木瓜样蛋白酶对IRF3信号通路的调节机制
发布时间：2018-12-24 12:04
【摘要】：天然免疫系统是宿主抵御病毒感染的第一道防线。宿主细胞通过病原相关分子模式（PAMPs）与其受体相互识别来激活天然免疫应答，诱导I型干扰素、促炎性因子等抗病毒活性分子的产生。冠状病毒是感染人类呼吸道的一类重要病毒。SARS，NL63等人类新发冠状病毒在进化过程中形成了特定机制逃避宿主天然免疫。SARS冠状病毒编码蛋白中，已发现多种蛋白（NSP1，PLpro，NSP7，NSP15，ORF3b，ORF6，M蛋白和N蛋白）具有抗病毒天然免疫活性。其中木瓜样蛋白酶（Papain-like protease，PLpro）能够抑制IRF3信号通路激活，阻止干扰素表达。但是，目前对SARS等人类新发冠状病毒PLpro调节宿主细胞IRF3信号通路的分子机制还不清楚。 PLpro是SARS冠状病毒编码的非结构蛋白NSP3中的一个蛋白酶催化结构域，我们前期研究发现PLpro具有去泛素化酶（Deubiquitinase，DUB）活性，而且PLpro下游的跨膜结构域（TM）对其功能有重要影响。本课题以带跨膜结构域的木瓜样蛋白酶PLpro-TM为研究对象，研究其对IRF3信号通路负调控的分子机制，研究发现：（一）通过报告基因检测技术发现PLpro-TM对RIG-I等调节蛋白介导的IRF3信号通路激活具有负调节作用；（二）利用免疫共沉淀技术检测发现PLpro-TM可以破坏E3泛素连接酶TRIM25与RIG-I CARD结构域的相互作用，同时对RIG-I具有去泛素化作用从而抑制RIG-I的活化，然而以上过程对IRF3信号通路的激活没有显著影响；（三） PLpro-TM能够通过破坏MAVS与TRAF3以及MAVS与STING间相互作用从而影响信号传递，但这种破坏并不能有效阻止IRF3活化；（四） PLpro-TM可以通过去除TRAF3连接的K63多聚泛素化修饰使其从信号复合体上解离，从而抑制IRF3活化；（五）通过非变性凝胶实验和磷酸化检测实验表明PLpro-TM能抑制STING诱导的IRF3磷酸化与二聚化，PLpro-TM能破坏STING与IRF3的相互作用抑制TBK1磷酸化IRF3，PLpro-TM还能与STING的TM结构域相互作用，可能借此抑制STING的移位从而阻断IRF3通路激活。总之，本课题发现了PLpro-TM负调节IRF3信号通路的两个作用位点TRAF3与STING，阐明了SARS等人类新发冠状病毒拮抗宿主抗病毒天然中IRF3信号通路调节的一种新机制，，为SARS等人类新发冠状病毒新型药物和疫苗研究提供重要理论基础。
[Abstract]:The innate immune system is the host's first line of defense against viral infection. The host cells activate the innate immune response by recognizing the pathogen-associated molecular model (PAMPs) and their receptors and induce the production of anti-viral molecules such as interferon type I and pro-inflammatory factors. Coronavirus is a kind of important virus that infects human respiratory tract. New human coronavirus, such as SARS,NL63, has formed a special mechanism to evade host innate immunity during evolution. Many proteins (NSP1,PLpro,) have been found in the protein encoded by SARS coronavirus. NSP7,NSP15,ORF3b,ORF6,M protein and N protein have anti-viral innate immune activity. Papain like protease (Papain-like protease,PLpro) can inhibit the activation of IRF3 signaling pathway and inhibit the expression of interferon. However, the molecular mechanism of SARS and other human coronavirus PLpro regulating the host cell IRF3 signaling pathway is unclear. PLpro is a protease-catalyzed domain of NSP3, a non-structural protein encoded by SARS coronavirus. Our previous studies have found that PLpro has the activity of Deubiquitinase,DUB, and the transmembrane domain (TM) downstream of PLpro has an important effect on its function. In this study, papain like protease PLpro-TM with transmembrane domain was used to study the molecular mechanism of its negative regulation on IRF3 signaling pathway. The results were as follows: (1) through reporter gene detection, PLpro-TM was found to have a negative effect on the activation of IRF3 signaling pathway mediated by RIG-I and other regulatory proteins; (2) using immunoprecipitation technique, it was found that PLpro-TM could destroy the interaction between the E3 ubiquitin ligase TRIM25 and the RIG-I CARD domain, and at the same time, it could devitalize the RIG-I and inhibit the activation of RIG-I. However, the above processes have no significant effect on the activation of IRF3 signaling pathway. (3) PLpro-TM can affect signal transduction by destroying the interaction between MAVS and TRAF3 and MAVS and STING, but this kind of destruction can not effectively prevent IRF3 activation. (4) PLpro-TM can inhibit the activation of IRF3 by removing the K63 polyubiquitin modification of TRAF3 binding and dissociating it from the signal complex; (5) PLpro-TM can inhibit IRF3 phosphorylation and dimerization induced by STING, and PLpro-TM can destroy the interaction between STING and IRF3 and inhibit TBK1 phosphorylation IRF3, by non-denaturing gel experiment and phosphorylation test. PLpro-TM can also interact with the TM domain of STING, which may inhibit the translocation of STING and block the activation of IRF3 pathway. In conclusion, TRAF3 and STING, two sites of PLpro-TM negatively regulating IRF3 signaling pathway, have demonstrated a new mechanism of IRF3 signal pathway regulation in human coronavirus antagonism against host virus, such as SARS. It provides an important theoretical basis for the study of new drugs and vaccines of human coronavirus such as SARS.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
本文编号：2390596
[Abstract]:The innate immune system is the host's first line of defense against viral infection. The host cells activate the innate immune response by recognizing the pathogen-associated molecular model (PAMPs) and their receptors and induce the production of anti-viral molecules such as interferon type I and pro-inflammatory factors. Coronavirus is a kind of important virus that infects human respiratory tract. New human coronavirus, such as SARS,NL63, has formed a special mechanism to evade host innate immunity during evolution. Many proteins (NSP1,PLpro,) have been found in the protein encoded by SARS coronavirus. NSP7,NSP15,ORF3b,ORF6,M protein and N protein have anti-viral innate immune activity. Papain like protease (Papain-like protease,PLpro) can inhibit the activation of IRF3 signaling pathway and inhibit the expression of interferon. However, the molecular mechanism of SARS and other human coronavirus PLpro regulating the host cell IRF3 signaling pathway is unclear. PLpro is a protease-catalyzed domain of NSP3, a non-structural protein encoded by SARS coronavirus. Our previous studies have found that PLpro has the activity of Deubiquitinase,DUB, and the transmembrane domain (TM) downstream of PLpro has an important effect on its function. In this study, papain like protease PLpro-TM with transmembrane domain was used to study the molecular mechanism of its negative regulation on IRF3 signaling pathway. The results were as follows: (1) through reporter gene detection, PLpro-TM was found to have a negative effect on the activation of IRF3 signaling pathway mediated by RIG-I and other regulatory proteins; (2) using immunoprecipitation technique, it was found that PLpro-TM could destroy the interaction between the E3 ubiquitin ligase TRIM25 and the RIG-I CARD domain, and at the same time, it could devitalize the RIG-I and inhibit the activation of RIG-I. However, the above processes have no significant effect on the activation of IRF3 signaling pathway. (3) PLpro-TM can affect signal transduction by destroying the interaction between MAVS and TRAF3 and MAVS and STING, but this kind of destruction can not effectively prevent IRF3 activation. (4) PLpro-TM can inhibit the activation of IRF3 by removing the K63 polyubiquitin modification of TRAF3 binding and dissociating it from the signal complex; (5) PLpro-TM can inhibit IRF3 phosphorylation and dimerization induced by STING, and PLpro-TM can destroy the interaction between STING and IRF3 and inhibit TBK1 phosphorylation IRF3, by non-denaturing gel experiment and phosphorylation test. PLpro-TM can also interact with the TM domain of STING, which may inhibit the translocation of STING and block the activation of IRF3 pathway. In conclusion, TRAF3 and STING, two sites of PLpro-TM negatively regulating IRF3 signaling pathway, have demonstrated a new mechanism of IRF3 signal pathway regulation in human coronavirus antagonism against host virus, such as SARS. It provides an important theoretical basis for the study of new drugs and vaccines of human coronavirus such as SARS.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
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