猪繁殖与呼吸综合征病毒非结构蛋白Nsp4调控IFN-β的分子机制研究
发布时间：2019-05-24 01:24
【摘要】：猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种病毒性传染病,给全球的养猪业造成了巨大的经济影响。该病主要临床症状为引起妊娠母猪严重的繁殖障碍以及各年龄段猪的呼吸系统疾病。PRRSV能够通过许多途径逃避宿主的天然免疫,抑制I型干扰素的产生。病毒的一些非结构蛋白在抑制干扰素产生的过程中都起到了重要作用。Nsp4是一种3C样丝氨酸蛋白酶(3CLSP),能够水解多聚蛋白pp1a/pp1ab产生Nsp3~Nsp12十种成熟的非结构蛋白,但目前PRRSV Nsp4抑制I型干扰素产生的分子机制尚不清楚。本研究证实了PRRSV Nsp4作为一种多功能的蛋白能够通过切割接头分子MAVS和NEMO阻碍I型干扰素的表达。随后的研究发现,PRRSV Nsp4切割MAVS依赖于其本身的蛋白酶活性,而不依赖细胞凋亡途径和蛋白酶体途径的方式。PRRSV的蛋白水解酶切割MAVS和NEMO也揭示了PRRSV调节天然免疫的新机制。主要的研究内容如下:1.PRRSV Nsp4通过阻碍IRF3和NF-κB的活化抑制RIG-I/MDA5介导的IFN-β的激活通过荧光素酶报告系统证实PRRSV Nsp4能够抑制小鼠仙台病毒(SEV)诱导IFN-β的表达。之后我们进一步证实PRRSV Nsp4通过阻碍IRF3和NF-κB的活化抑制RIG-I/MDA5介导的IFN-β的激活,且PRRSV Nsp4抑制IFN-β的产生呈剂量依赖性和酶活性依赖性。2.PRRSV Nsp4通过切割MAVS和NEMO抑制IFN-β的产生Nsp4是PRRSV主要的蛋白水解酶,在病毒的复制过程中起着重要作用,而PRRSV Nsp4抑制IFN-β的产生也呈现出酶活性依赖性。为了研究PRRSV Nsp4是否通过与RIG-I信号通路中的接头分子相互作用来抑制IFN-β的产生,将MAVS或NEMO真核表达质粒与Nsp4真核表达质粒共转染HEK-293T细胞,通过western-blot检测发现PRRSV Nsp4能够同时切割信号分子MAVS和NEMO,且呈酶活性依赖性。之后我们对PRRSV Nsp4切割MAVS做了进一步研究,发现Nsp4切割MAVS依赖于其自身的蛋白酶活性,而不依赖细胞凋亡及蛋白酶体途径。3.PRRSV Nsp4切割MAVS位点的确定研究表明,PRRSV Nsp4底物的酶切位点有一定的特异性,即PRRSV Nsp4通过识别底物Glu-Gly、Glu-Ala或Glu-Ser残基位点特异性切割底物。为了确定PRRSV Nsp4切割MAVS的位点,通过定点突变构建了MAVS的突变体真核表达质粒(MAVS-E268A、MAVS-E274A、MAVS-E288A),与Nsp4真核表达质粒共转染HEK-293T后,通过western blot检测发现PRRSV Nsp4不能切割MAVS-E268A突变体,说明PRRSV Nsp4通过识别MAVS第268位谷氨酸切割MAVS。与全长的MAVS相比,切割MAVS产生的两个片段(1-268和269-540)都不能够有效的诱导IFN-β的表达。
[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，
本文编号：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，
本文编号：2484425
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