豬繁殖與呼吸綜合征病毒非結(jié)構(gòu)蛋白Nsp4調(diào)控IFN-β的分子機制研究
發(fā)布時間：2019-05-24 01:24
【摘要】：豬繁殖與呼吸綜合征(PRRS)是由豬繁殖與呼吸綜合征病毒(PRRSV)引起的一種病毒性傳染病,給全球的養(yǎng)豬業(yè)造成了巨大的經(jīng)濟影響。該病主要臨床癥狀為引起妊娠母豬嚴(yán)重的繁殖障礙以及各年齡段豬的呼吸系統(tǒng)疾病。PRRSV能夠通過許多途徑逃避宿主的天然免疫,抑制I型干擾素的產(chǎn)生。病毒的一些非結(jié)構(gòu)蛋白在抑制干擾素產(chǎn)生的過程中都起到了重要作用。Nsp4是一種3C樣絲氨酸蛋白酶(3CLSP),能夠水解多聚蛋白pp1a/pp1ab產(chǎn)生Nsp3~Nsp12十種成熟的非結(jié)構(gòu)蛋白,但目前PRRSV Nsp4抑制I型干擾素產(chǎn)生的分子機制尚不清楚。本研究證實了PRRSV Nsp4作為一種多功能的蛋白能夠通過切割接頭分子MAVS和NEMO阻礙I型干擾素的表達(dá)。隨后的研究發(fā)現(xiàn),PRRSV Nsp4切割MAVS依賴于其本身的蛋白酶活性,而不依賴細(xì)胞凋亡途徑和蛋白酶體途徑的方式。PRRSV的蛋白水解酶切割MAVS和NEMO也揭示了PRRSV調(diào)節(jié)天然免疫的新機制。主要的研究內(nèi)容如下:1.PRRSV Nsp4通過阻礙IRF3和NF-κB的活化抑制RIG-I/MDA5介導(dǎo)的IFN-β的激活通過熒光素酶報告系統(tǒng)證實PRRSV Nsp4能夠抑制小鼠仙臺病毒(SEV)誘導(dǎo)IFN-β的表達(dá)。之后我們進一步證實PRRSV Nsp4通過阻礙IRF3和NF-κB的活化抑制RIG-I/MDA5介導(dǎo)的IFN-β的激活,且PRRSV Nsp4抑制IFN-β的產(chǎn)生呈劑量依賴性和酶活性依賴性。2.PRRSV Nsp4通過切割MAVS和NEMO抑制IFN-β的產(chǎn)生Nsp4是PRRSV主要的蛋白水解酶,在病毒的復(fù)制過程中起著重要作用,而PRRSV Nsp4抑制IFN-β的產(chǎn)生也呈現(xiàn)出酶活性依賴性。為了研究PRRSV Nsp4是否通過與RIG-I信號通路中的接頭分子相互作用來抑制IFN-β的產(chǎn)生,將MAVS或NEMO真核表達(dá)質(zhì)粒與Nsp4真核表達(dá)質(zhì)粒共轉(zhuǎn)染HEK-293T細(xì)胞,通過western-blot檢測發(fā)現(xiàn)PRRSV Nsp4能夠同時切割信號分子MAVS和NEMO,且呈酶活性依賴性。之后我們對PRRSV Nsp4切割MAVS做了進一步研究,發(fā)現(xiàn)Nsp4切割MAVS依賴于其自身的蛋白酶活性,而不依賴細(xì)胞凋亡及蛋白酶體途徑。3.PRRSV Nsp4切割MAVS位點的確定研究表明,PRRSV Nsp4底物的酶切位點有一定的特異性,即PRRSV Nsp4通過識別底物Glu-Gly、Glu-Ala或Glu-Ser殘基位點特異性切割底物。為了確定PRRSV Nsp4切割MAVS的位點,通過定點突變構(gòu)建了MAVS的突變體真核表達(dá)質(zhì)粒(MAVS-E268A、MAVS-E274A、MAVS-E288A),與Nsp4真核表達(dá)質(zhì)粒共轉(zhuǎn)染HEK-293T后,通過western blot檢測發(fā)現(xiàn)PRRSV Nsp4不能切割MAVS-E268A突變體,說明PRRSV Nsp4通過識別MAVS第268位谷氨酸切割MAVS。與全長的MAVS相比,切割MAVS產(chǎn)生的兩個片段(1-268和269-540)都不能夠有效的誘導(dǎo)IFN-β的表達(dá)。
[Abstract]:Pig Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome virus (PRRSV), which has a great economic impact on the global pig industry. The main clinical symptoms of the disease are severe reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. PRRSv can escape the innate immunity of the host through many ways and inhibit the production of type I interferon. Some non-structural proteins of the virus play an important role in inhibiting interferon production. Nsp4 is a 3C-like serine protease (3CLSP), which can hydrolyze polyprotein pp1a/pp1ab to produce ten mature non-structural proteins of Nsp3~Nsp12. However, the molecular mechanism of PRRSV Nsp4 inhibiting interferon type I production is not clear. This study confirmed that PRRSV Nsp4, as a multifunctional protein, can block the expression of interferon I by cutting junction molecules MAVS and NEMO. Subsequently, it was found that PRRSV Nsp4 cleaving MAVS depended on its own protease activity, but not on apoptosis pathway and proteasome pathway. PRRSv proteolytic enzyme cleaved MAVS and NEMO also revealed a new mechanism of PRRSV regulating innate immunity. The main research contents are as follows: 1.PRRSV Nsp4 can inhibit the activation of RIG-I/MDA5-mediated IFN- 尾 by blocking the activation of IRF3 and NF- 魏 B by luciferase reporting system. PRRSV Nsp4 can inhibit the induction of mouse Sendai virus (SEV) by luciferase reporting system. To induce the expression of interferon-尾. Then we further confirmed that PRRSV Nsp4 inhibited the activation of IFN- 尾 mediated by RIG-I/MDA5 by blocking the activation of IRF3 and NF- 魏 B. The inhibition of IFN- 尾 production by PRRSV Nsp4 was dose-dependent and enzyme activity dependent. 2. PRRSv Nsp4 inhibited the production of IFN- 尾 by cutting MAVS and NEMO, which is the main proteolytic enzyme of PRRSV and plays an important role in the process of virus replication. The inhibitory effect of PRRSV Nsp4 on the production of IFN- 尾 was also dependent on enzyme activity. In order to study whether PRRSV Nsp4 can inhibit the production of IFN- 尾 by interacting with junction molecules in RIG-I signaling pathway, MAVS or NEMO eukaryotic expression plasmid and Nsp4 eukaryotic expression plasmid are co-transfected into HEK-293T cells. By western-blot, it was found that PRRSV Nsp4 could cut both signal molecules MAVS and NEMO, in a enzyme activity dependent manner. After that, we further studied the cutting of MAVS by PRRSV Nsp4, and found that Nsp4 cutting MAVS depended on its own protease activity, but not on apoptosis and proteasome pathway. 3. The determination of MAVS site by PRRSv Nsp4 digestion showed that Nsp4 cutting MAVS depended on its own protease activity, but not on apoptosis and proteasome pathway. The restriction site of PRRSV Nsp4 substrate has certain specificity, that is, PRRSV Nsp4 cleans the substrate specifically by recognizing the substrate Glu-Gly,Glu-Ala or Glu-Ser residue site. In order to determine the site of PRRSV Nsp4 cleaving MAVS, the eukaryotic expression plasmid (MAVS-E268A,MAVS-E274A,MAVS-E288A) of MAVS was constructed by site-directed mutation and co-transfected with Nsp4 eukaryotic expression plasmid into HEK-293T. It was found that PRRSV Nsp4 could not cut MAVS-E268A mutant by western blot, which indicated that PRRSV Nsp4 could cleave MAVS. by recognizing glutamic acid at position 268 of MAVS. Compared with the full-length MAVS, the expression of IFN- 尾 could not be effectively induced by cutting the two fragments produced by MAVS (1 鈮，
本文編號：2484425
[Abstract]:Pig Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease caused by Porcine Reproductive and Respiratory Syndrome virus (PRRSV), which has a great economic impact on the global pig industry. The main clinical symptoms of the disease are severe reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. PRRSv can escape the innate immunity of the host through many ways and inhibit the production of type I interferon. Some non-structural proteins of the virus play an important role in inhibiting interferon production. Nsp4 is a 3C-like serine protease (3CLSP), which can hydrolyze polyprotein pp1a/pp1ab to produce ten mature non-structural proteins of Nsp3~Nsp12. However, the molecular mechanism of PRRSV Nsp4 inhibiting interferon type I production is not clear. This study confirmed that PRRSV Nsp4, as a multifunctional protein, can block the expression of interferon I by cutting junction molecules MAVS and NEMO. Subsequently, it was found that PRRSV Nsp4 cleaving MAVS depended on its own protease activity, but not on apoptosis pathway and proteasome pathway. PRRSv proteolytic enzyme cleaved MAVS and NEMO also revealed a new mechanism of PRRSV regulating innate immunity. The main research contents are as follows: 1.PRRSV Nsp4 can inhibit the activation of RIG-I/MDA5-mediated IFN- 尾 by blocking the activation of IRF3 and NF- 魏 B by luciferase reporting system. PRRSV Nsp4 can inhibit the induction of mouse Sendai virus (SEV) by luciferase reporting system. To induce the expression of interferon-尾. Then we further confirmed that PRRSV Nsp4 inhibited the activation of IFN- 尾 mediated by RIG-I/MDA5 by blocking the activation of IRF3 and NF- 魏 B. The inhibition of IFN- 尾 production by PRRSV Nsp4 was dose-dependent and enzyme activity dependent. 2. PRRSv Nsp4 inhibited the production of IFN- 尾 by cutting MAVS and NEMO, which is the main proteolytic enzyme of PRRSV and plays an important role in the process of virus replication. The inhibitory effect of PRRSV Nsp4 on the production of IFN- 尾 was also dependent on enzyme activity. In order to study whether PRRSV Nsp4 can inhibit the production of IFN- 尾 by interacting with junction molecules in RIG-I signaling pathway, MAVS or NEMO eukaryotic expression plasmid and Nsp4 eukaryotic expression plasmid are co-transfected into HEK-293T cells. By western-blot, it was found that PRRSV Nsp4 could cut both signal molecules MAVS and NEMO, in a enzyme activity dependent manner. After that, we further studied the cutting of MAVS by PRRSV Nsp4, and found that Nsp4 cutting MAVS depended on its own protease activity, but not on apoptosis and proteasome pathway. 3. The determination of MAVS site by PRRSv Nsp4 digestion showed that Nsp4 cutting MAVS depended on its own protease activity, but not on apoptosis and proteasome pathway. The restriction site of PRRSV Nsp4 substrate has certain specificity, that is, PRRSV Nsp4 cleans the substrate specifically by recognizing the substrate Glu-Gly,Glu-Ala or Glu-Ser residue site. In order to determine the site of PRRSV Nsp4 cleaving MAVS, the eukaryotic expression plasmid (MAVS-E268A,MAVS-E274A,MAVS-E288A) of MAVS was constructed by site-directed mutation and co-transfected with Nsp4 eukaryotic expression plasmid into HEK-293T. It was found that PRRSV Nsp4 could not cut MAVS-E268A mutant by western blot, which indicated that PRRSV Nsp4 could cleave MAVS. by recognizing glutamic acid at position 268 of MAVS. Compared with the full-length MAVS, the expression of IFN- 尾 could not be effectively induced by cutting the two fragments produced by MAVS (1 鈮，
本文編號：2484425
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