猪繁殖与呼吸综合征病毒N蛋白SUMO化修饰对病毒复制及N蛋白功能的影响

发布时间：2018-06-23 03:27

本文选题：猪繁殖与呼吸综合征病毒 + 核衣壳蛋白　；参考：《中国农业大学》2017年博士论文

【摘要】：猪繁殖与呼吸综合征病毒(PRRSV)是危害世界养猪生产的重要病原,并造成巨大的经济损失。其核衣壳蛋白(N蛋白)是病毒粒子重要的内部结构蛋白及免疫原性蛋白,参与病毒RNA合成及感染性病毒颗粒装配,且N蛋白核定位(Nuclear Localization Signal, NLS)与PRRSV致病性密切相关。SUMO是一类重要的类泛素蛋白,广泛参与生物体生命活动,病毒蛋白的SUMO化修饰在抑制宿主先天性免疫、参与病毒免疫逃逸及复制中发挥重要作用。本论文围绕PRRSV N蛋白SUMO化修饰对病毒体外复制、N蛋白亚细胞定位和抑制β干扰素产生的影响开展研究,旨在从蛋白翻译后修饰的新视角为PRRSV致病机制研究提供线索,也为抗PRRSV药物研发提供新靶点。为了验证PRRSV蛋白的SUMO化修饰,利用分析软件SUMOSp1.0a、SUMOplot和seeSUMO进行预测,并以SUMOE2结合酶Ubc9蛋白作为“诱饵”蛋白,采用酵母回复杂交技术筛选,发现Ubc9与PRRSV的Nsp1β、Nsp4、Nsp9、Nsp10及N存在相互作用。进一步利用免疫共沉淀(Co-IP)、GST pull-down和激光共聚焦技术证实Ubc9与上述蛋白存在互作。为了分析Ubc9对PRRSV体外复制和基因组RNA合成的影响,利用RNA干扰和慢病毒包装技术分别沉默和过表达MARC-145细胞中Ubc9,结果显示,MARC-145细胞中过表达Ubc9在24 h可显著抑制PRRSV复制(尸0.01)及在24 h和36 h显著抑制PRRSV基因组RNA的合成(P0.001)。最后,为了分析SUMO化抑制剂是否有利于PRRSV复制,银杏酸(Ginkgolic acid, GA)处理MARC-145细胞4 h,数据显示,PRRSV在12 h和24 h于GA处理后的MARC-145细胞中的复制能力显著高于对照组。为了阐明PRRSV N蛋白SUMO化修饰的途径,利用定点突变技术对N蛋白中的赖氨酸进行组合突变,发现只有当N蛋白中的赖氨酸全部突变为精氨酸时,SUMO化修饰现象才消失,突变其任何一点或多点赖氨酸N蛋白仍能发生SUMO化修饰,由此证明N蛋白赖氨酸对其SUMO 化修饰是冗余的。PIAS1 (Protein inhibitor of activated STAT1)作为一种 SUMO E3 连接酶,可以促进靶蛋白的SUMO化修饰,进而影响靶蛋白的功能,参与基因转录调控过程。本研究利用SUMO化修饰体外检测试剂盒和免疫共沉淀技术对N蛋白SUMO修饰途径进行鉴定,验证PIAS1与N蛋白的相互作用以及分析PIAS1在N蛋白SUMO化修饰及PRRSV复制中的作用。研究结果表明,PIAS1能与N蛋白相互作用,两者共定位于胞浆中;外源转染PIAS1不能增加N蛋白SUMO化修饰水平;在MARC-145细胞中,PIAS1的表达有利于PRRSV的复制。鉴于赖氨酸对N蛋白SUMO化修饰是冗余的,本研究构建了系列N蛋白赖氨酸突变体并拯救病毒,进一步验证N蛋白中赖氨酸与其亚细胞定位、干扰素产生和PRRSV复制之间的相关性。基于赖氨酸在N蛋白中的分布,以pCMV-HA-N为模板,利用定点突变技术构建出系列N蛋白赖氨酸突变体;同时,以PRRSV高致病性毒株JXwn06的感染性cDNA克隆(pWSK-JXwn)为骨架,定点突变N蛋白中的赖氨酸,共拯救出6株N蛋白赖氨酸突变病毒,分别命名为RvJXwn、RvJXNK7,28,39,52R、RvJXNmutNLS1、RvJXNmutNLS2、RvJXNmutNLS1,2 和 RvJXNKR。分析突变体拯救病毒在MARC-145细胞和原代PAMs细胞上的增殖动态,发现当位于N蛋白NLS1、NLS2、NLS1,2上的赖氨酸及N蛋白中的全部赖氨酸突变为精氨酸后,突变体拯救病毒的体外增殖能力显著低于亲本病毒RvJXwn。利用激光共聚焦方法观察N蛋白赖氨酸突变体及突变体拯救病毒的亚细胞定位,结果表明,野生型N蛋白和NmutNLS2位于细胞核核仁,而其余N蛋白赖氨酸突变体则分布在细胞核除核仁外的核质中;突变体拯救病毒N蛋白则同亲本病毒一样分布于细胞核核仁和胞浆中。利用双荧光素试验分析N蛋白及其赖氨酸突变体对IFN-β和IRFs启动子活性的影响,证实NK7,28,39,52R和野生型N蛋白一样可以抑制IFN-β和IRFs启动子活性,而其余N蛋白赖氨酸突变体对IFN-β和IRFs启动子活性的抑制能力明显减弱。针对突变体拯救病毒在MARC-145细胞和原代PAMs细胞诱导产生IFN-β能力的检测结果表明,不同突变体拯救病毒感染宿主细胞诱导产生IFN-β的能力有明显差异,其中RvJXNmutNLS1、RvJXNmutNLS2、RvJXNmutNLS1,2 和 RvJXNKR 在感染后 24 h 和 36h诱导产生 IFN-β 水平极显著高于亲本病毒RvJXwn(P0.001 ),而RvJXNK7,28,39,52R与亲本病毒差异不显著(P0.05)。综上,本研究验证了 PRRSVNsp1β、Nsp4、Nsp9、Nsp10及N蛋白与宿主细胞蛋白Ubc9存在相互作用,证实了 PRRSVN蛋白存在SUMO化修饰现象,并确定赖氨酸对N蛋白SUMO化修饰是冗余的。通过构建N蛋白赖氨酸突变体及拯救其突变病毒,证实N蛋白赖氨酸突变体可影响IFN-β和IRFs启动子活性及改变N蛋白的亚细胞定位;除第7,28,39,52位赖氨酸外,N蛋白其余赖氨酸与病毒的体外增殖能力密切相关,且与诱导IFN-β水平有关。
[Abstract]:Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that endangers the production of swine in the world and causes huge economic losses. Its nucleocapsid protein (N protein) is an important internal structural protein and immunogenic protein of virus particles, and is involved in the synthesis of virus RNA and the assembly of infected venereal particles, and the localization of N protein nuclei (Nuclear Localization S). Ignal, NLS), which is closely related to the pathogenicity of PRRSV,.SUMO is an important class of ubiquitin protein, which is widely involved in biological activities. The SUMO modification of virus protein plays an important role in inhibiting the host's innate immunity and participating in the immune escape and replication of the virus. This paper revolves around the SUMO modification of the PRRSV N protein in vitro and N protein. The effects of subcellular localization and inhibition of interferon production were studied. The aim of the study was to provide clues for the study of the pathogenesis of PRRSV, and to provide new targets for the research and development of anti PRRSV drugs. In order to verify the SUMO modification of PRRSV protein, the analysis software SUMOSp1.0a, SUMOplot and seeSUMO were used to predict, and SUMOE2 was used. Combining enzyme Ubc9 protein as bait protein, the interaction between Ubc9 and Nsp1 beta, Nsp4, Nsp9, Nsp10 and N was found by yeast recovery hybridization. The interaction of Ubc9 with the above protein was confirmed by immunoprecipitation (Co-IP), GST pull-down and laser confocal technology. The effect of genomic RNA synthesis, using RNA interference and lentivirus packaging techniques to silence and overexpress Ubc9 in MARC-145 cells, the results show that overexpression of Ubc9 in 24 h can significantly inhibit PRRSV replication (corpse 0.01) and significantly inhibit the synthesis of PRRSV genomic RNA in 24 h and 36 h. Finally, in order to analyze the inhibitors Whether it was beneficial to PRRSV replication, Ginkgolic acid (GA) was used to treat MARC-145 cells 4 h. The data showed that the replication ability of PRRSV in MARC-145 cells treated with 12 h and 24 h was significantly higher than that of the control group. It was found that only when the lysine in the N protein suddenly mutated to arginine, the SUMO modification disappeared, and any point or multiple point of the lysine N protein could still be modified by SUMO, which proved that the SUMO modification of N protein lysine was a redundant.PIAS1 (Protein inhibitor of activated STAT1) as a kind of connection. Enzyme, which can promote the SUMO modification of target protein, and then influence the function of target protein and participate in the process of gene transcription regulation. This study uses SUMO modified in vitro detection kit and immunoprecipitation technology to identify the N protein SUMO modification pathway, and verify the interaction between PIAS1 and N protein and analyze the PIAS1 in the SUMO modification of N protein and PRRS. The effect of V replication. The results show that PIAS1 can interact with N protein and both are located in the cytoplasm; exogenous transfection of PIAS1 can not increase the SUMO modification level of N protein; in MARC-145 cells, the expression of PIAS1 is beneficial to the replication of PRRSV. In view of lysine, the SUMO modification of N protein is redundant. This study constructs a series of N protein Reys. The amino acid mutant was used to save the virus and further verify the correlation between lysine and its subcellular localization, interferon production and PRRSV replication in N protein. Based on the distribution of lysine in N protein, a series of N protein lysine mutants were constructed by pCMV-HA-N as a template, and the PRRSV highly pathogenic strain JXwn06 was also constructed. The infectious cDNA clone (pWSK-JXwn) was the skeleton, and the lysine in the fixed-point mutation of N protein saved 6 N protein lysine mutant viruses, which were named RvJXwn, RvJXNK7,28,39,52R, RvJXNmutNLS1, RvJXNmutNLS2, RvJXNmutNLS1,2 and RvJXNKR. analysis mutants to save the proliferation of the virus in MARC-145 cells and primary PAMs cells. It was found that when the lysine and all lysine in the N protein NLS1, NLS2, NLS1,2 and N protein were mutated to arginine, the ability of the mutant to save the virus in vitro was significantly lower than the parent virus RvJXwn. using the laser confocal method to observe the subcellular localization of the N protein lysine mutant and the mutant rescue virus. The results showed that the wild virus was located in the wild. The N protein and NmutNLS2 are located in nucleus nucleolus, while the other N protein lysine mutants are distributed in nucleus except nucleolus. The mutant rescue virus N protein distributes in nucleus nucleolus and cytoplasm like the parent virus. Using the double fluorescein test, the N protein and its lysine mutant are analyzed for IFN- beta and IRFs promoter. The effect of activity, confirmed that NK7,28,39,52R and wild type N protein can inhibit the activity of IFN- beta and IRFs promoter, while the inhibition ability of the other N protein lysine mutants on the activity of IFN- beta and IRFs promoters decreased obviously. There were significant differences in the ability to induce IFN- beta induced by different mutants to save the virus infected host cells, in which RvJXNmutNLS1, RvJXNmutNLS2, RvJXNmutNLS1,2 and RvJXNKR were significantly higher than parental virus RvJXwn (P0.001) induced by 24 h and 36h after infection (P0.001), while RvJXNK7,28,39,52R was not significantly different from parental virus (P0.). 05) 05) to sum up, this study verified the interaction of PRRSVNsp1 beta, Nsp4, Nsp9, Nsp10 and N protein with the host cell protein Ubc9, which confirmed the SUMO modification of the PRRSVN protein and determined that lysine was redundant to the N protein SUMO modification. The N protein lysine was confirmed by the construction of the N protein lysine mutant and the rescue of the mutant virus. The mutants can affect the activity of IFN- beta and IRFs promoters and change the subcellular localization of N proteins. Except for the 7,28,39,52 site lysine, the remaining lysine of the N protein is closely related to the proliferation ability of the virus in vitro, and is related to the level of the induced IFN- beta.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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