SARS冠狀病毒木瓜樣蛋白酶對IRF3信號通路的調(diào)節(jié)機制
發(fā)布時間：2018-12-24 12:04
【摘要】：天然免疫系統(tǒng)是宿主抵御病毒感染的第一道防線。宿主細胞通過病原相關(guān)分子模式（PAMPs）與其受體相互識別來激活天然免疫應答，誘導I型干擾素、促炎性因子等抗病毒活性分子的產(chǎn)生。冠狀病毒是感染人類呼吸道的一類重要病毒。SARS，NL63等人類新發(fā)冠狀病毒在進化過程中形成了特定機制逃避宿主天然免疫。SARS冠狀病毒編碼蛋白中，已發(fā)現(xiàn)多種蛋白（NSP1，PLpro，NSP7，NSP15，ORF3b，ORF6，M蛋白和N蛋白）具有抗病毒天然免疫活性。其中木瓜樣蛋白酶（Papain-like protease，PLpro）能夠抑制IRF3信號通路激活，阻止干擾素表達。但是，目前對SARS等人類新發(fā)冠狀病毒PLpro調(diào)節(jié)宿主細胞IRF3信號通路的分子機制還不清楚。 PLpro是SARS冠狀病毒編碼的非結(jié)構(gòu)蛋白NSP3中的一個蛋白酶催化結(jié)構(gòu)域，我們前期研究發(fā)現(xiàn)PLpro具有去泛素化酶（Deubiquitinase，DUB）活性，而且PLpro下游的跨膜結(jié)構(gòu)域（TM）對其功能有重要影響。本課題以帶跨膜結(jié)構(gòu)域的木瓜樣蛋白酶PLpro-TM為研究對象，研究其對IRF3信號通路負調(diào)控的分子機制，研究發(fā)現(xiàn)：（一）通過報告基因檢測技術(shù)發(fā)現(xiàn)PLpro-TM對RIG-I等調(diào)節(jié)蛋白介導的IRF3信號通路激活具有負調(diào)節(jié)作用；（二）利用免疫共沉淀技術(shù)檢測發(fā)現(xiàn)PLpro-TM可以破壞E3泛素連接酶TRIM25與RIG-I CARD結(jié)構(gòu)域的相互作用，同時對RIG-I具有去泛素化作用從而抑制RIG-I的活化，然而以上過程對IRF3信號通路的激活沒有顯著影響；（三） PLpro-TM能夠通過破壞MAVS與TRAF3以及MAVS與STING間相互作用從而影響信號傳遞，但這種破壞并不能有效阻止IRF3活化；（四） PLpro-TM可以通過去除TRAF3連接的K63多聚泛素化修飾使其從信號復合體上解離，從而抑制IRF3活化；（五）通過非變性凝膠實驗和磷酸化檢測實驗表明PLpro-TM能抑制STING誘導的IRF3磷酸化與二聚化，PLpro-TM能破壞STING與IRF3的相互作用抑制TBK1磷酸化IRF3，PLpro-TM還能與STING的TM結(jié)構(gòu)域相互作用，可能借此抑制STING的移位從而阻斷IRF3通路激活。總之，本課題發(fā)現(xiàn)了PLpro-TM負調(diào)節(jié)IRF3信號通路的兩個作用位點TRAF3與STING，闡明了SARS等人類新發(fā)冠狀病毒拮抗宿主抗病毒天然中IRF3信號通路調(diào)節(jié)的一種新機制，，為SARS等人類新發(fā)冠狀病毒新型藥物和疫苗研究提供重要理論基礎。
[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.
【學位授予單位】：北京工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
本文編號：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.
【學位授予單位】：北京工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R373
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本文編號：2390596
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