EV71病毒抑制RIG-Ⅰ泛素化逃避宿主天然免疫的机制研究

发布时间：2018-01-15 00:01

本文关键词：EV71病毒抑制RIG-Ⅰ泛素化逃避宿主天然免疫的机制研究　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

更多相关文章： EV71 RIG-Ⅰ IFN-β 泛素化 天然免疫

【摘要】：肠道病毒71型(Enteroviruses 71,EV71)属于小核糖核酸病毒科,肠道病毒属,肠道病毒A型的成员,是引起婴幼儿手足口病的重要病原体。近年来,EV71病毒在全世界多次爆发与流行,造成数万人的死亡。EV71病毒侵入细胞之后,病毒基因组复制形成双链RNA分子,会被宿主细胞的模式识别受体RIG-Ⅰ识别,随后RIG-Ⅰ受体分子在泛素连接酶作用下被泛素化,从而启动下游I型干扰素信号通路。本课题组前期研究发现,EV71病毒感染可以抑制宿主RIG-Ⅰ受体的泛素化水平,但是抑制RIG-Ⅰ泛素化是否最终介导了EV71病毒逃避宿主天然免疫尚不清楚。因此,本论文将以这一发现为切入点,阐明EV71病毒抑制RIG-Ⅰ泛素化逃避宿主天然免疫这一科学问题,为EV71病毒逃避宿主天然免疫理论提出新的观点。为了研究EV71病毒在RD细胞内的复制情况,EV71病毒感染RD细胞24小时之后,采用光学显微镜和荧光显微镜在不同时间点观察细胞病变。结果显示,EV71病毒感染6小时,检测到大量病毒蛋白表达,且细胞出现明显病变。在确定感染时间点的基础上,细胞转染IFN-β-Luc质粒之后,采用双荧光素酶报告基因系统,检测EV71病毒感染对RD细胞IFN-β启动子活性的影响。结果显示,EV71(MOI=20)病毒显著降低RD细胞IFN-β的启动子活性。为了确认EV71病毒对IFN-β的抑制作用,采用RT-PCR方法检测病毒对IFN-β转录水平的影响。结果显示,EV71病毒感染显著抑制细胞IFN-β的转录。干扰素调节基因3(IRF3)的入核对于活化宿主天然免疫是必不可少的,为了证实EV71病毒通过抑制IRF3的入核拮抗I型干扰素通路的可能性,RD细胞转染pEGFP-IRF3质粒之后,采用免疫荧光技术观察IRF3的入核情况。结果显示,EV71病毒感染明显抑制了IRF3的入核。干扰素刺激基因(ISG)是发挥抗病毒作用的最终效应分子,为了研究EV71病毒对ISGs的影响,在EV71病毒感染RD细胞之后,采用RT-PCR方法检测病毒ISG54、ISG56以及IFN-β的转录水平,结果显示,EV71病毒感染显著下调RD细胞IFN-β、ISGs的mRNA转录水平。为了研究EV71病毒对RIG-Ⅰ受体泛素化的影响,EV71病毒感染RD细胞之后,采用Western Blot方法检测细胞内源性RIG-Ⅰ的泛素化水平。结果显示,EV71病毒感染显著抑制细胞内源性RIG-Ⅰ的泛素化,且表现为时间依赖性。为了证实EV71病毒对RIG-Ⅰ的抑制作用,RD细胞共转Flag-RIG-Ⅰ和HA-Ub质粒,采用pull down技术和Western Blot方法分析异源表达RIG-Ⅰ的泛素化水平,结果显示,EV71病毒感染同样显著抑制细胞异源表达的RIG-Ⅰ泛素化。为了进一步确定RIG-Ⅰ泛素化的抑制类型,本研究将Flag-RIG-Ⅰ质粒分别与HA-Ub、HA-Ub K63或HA-Ub K48质粒共转染至RD细胞,采用免疫共沉淀技术,分析RIG-Ⅰ的泛素化抑制类型。结果显示,EV71病毒显著抑制RIG-Ⅰ的K63位泛素化,而并不抑制K48位的泛素化。随后,RD细胞共转染HA-Ub和Flag-RIG-Ⅰ质粒,采用过表达技术提升RIG-Ⅰ的泛素化水平,并检测IFN-β和ISGs mRNA的转录水平,结果显示,上调RIG-Ⅰ的泛素化水平可以显著增强IFN-β和ISGs的转录水平。上述研究结果说明,EV71病毒感染RD细胞之后,首先抑制宿主细胞RIG-Ⅰ受体K63位的泛素化,随后阻断IRF3的入核,抑制IFN-β的转录和表达,最终阻断了天然免疫效应分子ISG54、ISG56的转录和表达,从而逃避了宿主天然免疫对自身的清除。本研究不仅有助于丰富EV71病毒与宿主天然免疫相互作用的理论知识,而且有助于发现潜在的抗病毒复制的新策略。
[Abstract]:Enterovirus 71 (Enteroviruses 71, EV71) belongs to the Picornaviridae family members, enterovirus, enterovirus A, is an important pathogen of hand foot and mouth disease in children. In recent years, the outbreak of EV71 virus in the world and popular, causing tens of thousands of people died after.EV71 virus invades a cell, the virus replication of the genome forming a double stranded RNA molecule, RIG- I will be recognition receptor pattern recognition of host cells, then RIG- receptor molecules in the ubiquitin ligase by ubiquitination, thereby initiating downstream of type I interferon signaling pathway. Our previous studies showed that EV71 infection can inhibit host RIG- receptor ubiquitination level. But I will eventually inhibit RIG- ubiquitination mediated by the EV71 virus to evade host innate immunity is unclear. Therefore, this paper will be to this discovery as the starting point, to clarify the EV71 virus inhibition of RIG- I pan In the escape this scientific problem of host innate immunity, EV71 virus to evade the host innate immune theory put forward new ideas. In order to study the replication of EV71 virus in RD cells, after EV71 infected RD cells 24 hours, using the optical microscope and fluorescence microscope at different time points to observe cell lesions. The results showed that EV71 virus infection 6 hours, detected expression of viral proteins, and cell lesions. Based on determining the infection time points, after the beta cells transfected with IFN- -Luc plasmid by dual luciferase reporter assay system for detection of EV71 virus infection promoter activity in RD cells. The results showed that IFN- beta, EV71 (MOI=20) the virus RD IFN- beta cells significantly reduced promoter activity. In order to confirm the inhibitory effect of EV71 virus on IFN- beta, RT-PCR method is used to influence the detection of viruses on IFN- beta transcription level. The results showed that Transcription, EV71 infection significantly inhibited the cell IFN-. Beta interferon regulated genes 3 (IRF3) into the nucleus is essential for the activation of host innate immunity, in order to confirm the possibility of EV71 virus through inhibition of IRF3 into the nucleus of antagonistic type I interferon pathway, RD cells transfected with pEGFP-IRF3 plasmid by immunofluorescence staining with IRF3 the nuclear situation. The results showed that EV71 infection inhibited the IRF3 into the nucleus. Interferon stimulated gene (ISG) is the end effector molecules exert antiviral effect, in order to study the effect of EV71 virus on ISGs, after EV71 infected RD cells, virus detection using RT-PCR ISG54 method, ISG56 and IFN- beta mRNA. The results showed that EV71 infection significantly reduced RD cell IFN- beta, ISGs mRNA transcription level. In order to study the effect of EV71 virus on RIG- receptor ubiquitination, EV71 infected RD cells, The ubiquitination level using the Western Blot method to detect endogenous RIG- 1. The results showed that EV71 infection significantly inhibited the endogenous RIG- of ubiquitination, and showed a time dependent manner. In order to confirm the inhibitory effect of EV71 virus on RIG- I, Flag-RIG- I and HA-Ub plasmid co transfection of RD cells, the ubiquitin level by pull down technology and Western Blot analysis showed that heterologous expression of RIG- 1, EV71 virus infection also significantly inhibited the heterologous expression of RIG- 1 ubiquitination. In order to further determine the type of inhibition of RIG- I ubiquitination, this research will Flag-RIG- 1 and HA-Ub HA-Ub plasmid respectively, K63 or HA-Ub K48 plasmid were transfected into RD cells. Using CO immunoprecipitation techniques, analysis of RIG- I ubiquitination inhibition type. The results showed that EV71 significantly inhibited the virus of RIG- I K63 ubiquitination, but does not inhibit K48 ubiquitination then, RD cells were transfected with HA-Ub plasmid and Flag-RIG- I, by over expression of ubiquitin to enhance the level of RIG- technology, and the results show that the detection level of transcription, IFN- beta and ISGs mRNA, the ubiquitin upregulation of RIG- I can significantly enhance the transcription level of IFN- beta and ISGs. The results show that after EV71 virus infection first of all RD cells, inhibit host cell receptor RIG- K63 ubiquitination, then blocking IRF3 into the nucleus, inhibition of transcription and expression of IFN- beta, eventually blocking the natural immune effector molecule ISG54, and the expression of ISG56 transcription, so as to avoid the removal of host innate immunity to itself. The research has not only theoretical knowledge enrich the EV71 virus and the host immune interaction, but also help to find new strategies for potential virus replication.

【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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