猪繁殖与呼吸综合征病毒Nsp4与细胞色素C1相互作用及其诱导细胞凋亡的分子机制
发布时间：2019-06-25 19:39
【摘要】：猪繁殖与呼吸综合征病毒(PRRSV)是严重危害全球养猪生产的重要病原,高致病性PRRSV的出现和流行给我国养猪业造成了巨大的经济损失。PRRSV基因组可编码产生至少14个非结构蛋白,其中非结构蛋白4 (Nsp4)具有3C样丝氨酸蛋白酶活性,在病毒的复制、抑制宿主天然免疫反应和诱导宿主细胞凋亡的过程中具有重要作用。本研究以PRRSV非结构蛋白Nsp4为研究对象,筛选与鉴定出与PRRSV Nsp4相互作用的猪肺泡巨噬细胞蛋白—细胞色素c1(Cytochrome c1,Cyto.c1),进一步研究了其相互作用及其影响Nsp4诱导细胞凋亡的分子机制,以期揭示Nsp4新的生物学功能,并为阐明PRRSV的分子致病机制提供科学依据。利用PRRSV高致病性毒株JXwn06,采用慢病毒包装技术,成功构建并拯救插入JXwn06 Nsp4基因及其酶活位点单点突变的重组慢病毒,并成功侵染MARC-145细胞,最终获得表达融合蛋白的Nsp4单点突变体传代细胞系。由于Nsp4可诱导细胞凋亡,未能获得Nsp4稳定表达的细胞系。为研究Nsp4诱导细胞凋亡的分子机制,以Nsp4作为“诱饵”蛋白,利用猪肺泡巨噬细胞cDNA文库和酵母双杂交技术,成功筛选获得与凋亡调控和凋亡信号传导相关蛋白—细胞色素c1(Cyto.c1),并通过酵母回交试验加以确认。利用免疫共沉淀技术(Co-IP)和激光共聚焦试验验证了 Nsp4和Cyto.c1在宿主细胞中相互作用,并确定其相互作用的区域分别为Nsp4的N端(1～160aa)和 Cyto.c1 的 N 端(1～230aa)。利用siRNA技术沉默MARC-145细胞中内源性Cyto.c1表达,分析Nsp4与Cyto.c1相互作用对Nsp4诱导细胞凋亡的影响。结果表明,干扰Cyto.c1表达后,Nsp4所诱导的细胞凋亡被显著抑制。同时,发现在PRRSV感染和质粒转染的情况下,Nsp4可以剪切Cyto.c1,剪切能力是由其3C样丝氨酸蛋白酶活性所决定的,并呈现剂量依赖性。进一步研究发现,Cyto.c1的E230aa～G231aa是决定Nsp4剪切活性的关键位点。Cyto.c1被剪切后形成的Cyto.c1/1～230aa蛋白可明显激活Caspase-3,同时引起线粒体的片段化,进而导致线粒体损伤,激活线粒体凋亡通路,最后诱导细胞凋亡的发生。综上所述,本研究筛选并鉴定出PRRSV Nsp4可与宿主细胞蛋白Cyto.c1相互作用,揭示了这一相互作用在Nsp4诱导宿主细胞凋亡中的分子机制,为阐明PRRSV的致病机理提供了有价值的科学依据。
[Abstract]:Pig reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that seriously endangers the global pig production. The emergence and epidemic of highly pathogenic PRRSV has caused great economic losses to the pig industry in China. PRRSv genome can code to produce at least 14 non-structural proteins, among which non-structural protein 4 (Nsp4) has 3C-like serine protease activity and replication in the virus. Inhibition of host innate immune response and induction of host cell apoptosis play an important role. In this study, PRRSV non-structural protein Nsp4 was selected and identified to screen and identify porcine alveolar macrophage protein C1 (Cytochrome C1, Cyto.c1) interacting with Nsp4. The interaction and its molecular mechanism of Nsp4 induced apoptosis were further studied in order to reveal the new biological function of Nsp4 and provide scientific basis for the molecular pathogenesis of PRRSV. Using lentivirus packaging technique, PRRSV highly pathogenic strain JXwn06, successfully constructed and rescued the recombinant lentivirus inserted into JXwn06 Nsp4 gene and its enzyme activity site single point mutation, and successfully infected MARC-145 cells. Finally, the Nsp4 single point mutant passage cell line expressing fusion protein was obtained. Because Nsp4 can induce apoptosis, the cell line with stable expression of Nsp4 can not be obtained. In order to study the molecular mechanism of apoptosis induced by Nsp4, using Nsp4 as bait protein, the protein related to apoptosis regulation and apoptosis signal transduction, cytochrome C1 (Cyto.c1), was successfully screened by cDNA library and yeast two-hybrid technique, and confirmed by yeast backcross test. The interaction between Nsp4 and Cyto.c1 in host cells was verified by immunoprecipitation technique (Co-IP) and laser confocal assay, and the N-terminal (1~160aa) of Nsp4 and N-terminal (1~230aa) of Cyto.c1 were determined to interact with each other. The expression of endogenous Cyto.c1 in MARC-145 cells was silenced by siRNA technique, and the effect of the interaction between Nsp4 and Cyto.c1 on apoptosis induced by Nsp4 was analyzed. The results showed that the apoptosis induced by Nsp4 was significantly inhibited after interfering with the expression of Cyto.c1. At the same time, it was found that the shear ability of Nsp4 to shear Cyto.c1, was determined by the activity of 3C-like serine protease in a dose-dependent manner in the case of PRRSV infection and plasmid transfer. It is found that the E230aa~G231aa of Cyto.c1 is the key site to determine the shear activity of Nsp4. The Cyto.c1/1~230aa protein formed after Cyto.c1 is cut can obviously activate Caspase-3, and induce the fragmentation of mitochondria, which can lead to the damage of mitochondria, activate the apoptosis pathway of mitochondria, and finally induce the occurrence of apoptosis. In conclusion, the interaction between PRRSV Nsp4 and host cell protein Cyto.c1 was screened and identified in this study, which revealed the molecular mechanism of this interaction in Nsp4 induced apoptosis of host cells, and provided a valuable scientific basis for elucidating the pathogenic mechanism of PRRSV.
【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
本文编号：2505948
[Abstract]:Pig reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that seriously endangers the global pig production. The emergence and epidemic of highly pathogenic PRRSV has caused great economic losses to the pig industry in China. PRRSv genome can code to produce at least 14 non-structural proteins, among which non-structural protein 4 (Nsp4) has 3C-like serine protease activity and replication in the virus. Inhibition of host innate immune response and induction of host cell apoptosis play an important role. In this study, PRRSV non-structural protein Nsp4 was selected and identified to screen and identify porcine alveolar macrophage protein C1 (Cytochrome C1, Cyto.c1) interacting with Nsp4. The interaction and its molecular mechanism of Nsp4 induced apoptosis were further studied in order to reveal the new biological function of Nsp4 and provide scientific basis for the molecular pathogenesis of PRRSV. Using lentivirus packaging technique, PRRSV highly pathogenic strain JXwn06, successfully constructed and rescued the recombinant lentivirus inserted into JXwn06 Nsp4 gene and its enzyme activity site single point mutation, and successfully infected MARC-145 cells. Finally, the Nsp4 single point mutant passage cell line expressing fusion protein was obtained. Because Nsp4 can induce apoptosis, the cell line with stable expression of Nsp4 can not be obtained. In order to study the molecular mechanism of apoptosis induced by Nsp4, using Nsp4 as bait protein, the protein related to apoptosis regulation and apoptosis signal transduction, cytochrome C1 (Cyto.c1), was successfully screened by cDNA library and yeast two-hybrid technique, and confirmed by yeast backcross test. The interaction between Nsp4 and Cyto.c1 in host cells was verified by immunoprecipitation technique (Co-IP) and laser confocal assay, and the N-terminal (1~160aa) of Nsp4 and N-terminal (1~230aa) of Cyto.c1 were determined to interact with each other. The expression of endogenous Cyto.c1 in MARC-145 cells was silenced by siRNA technique, and the effect of the interaction between Nsp4 and Cyto.c1 on apoptosis induced by Nsp4 was analyzed. The results showed that the apoptosis induced by Nsp4 was significantly inhibited after interfering with the expression of Cyto.c1. At the same time, it was found that the shear ability of Nsp4 to shear Cyto.c1, was determined by the activity of 3C-like serine protease in a dose-dependent manner in the case of PRRSV infection and plasmid transfer. It is found that the E230aa~G231aa of Cyto.c1 is the key site to determine the shear activity of Nsp4. The Cyto.c1/1~230aa protein formed after Cyto.c1 is cut can obviously activate Caspase-3, and induce the fragmentation of mitochondria, which can lead to the damage of mitochondria, activate the apoptosis pathway of mitochondria, and finally induce the occurrence of apoptosis. In conclusion, the interaction between PRRSV Nsp4 and host cell protein Cyto.c1 was screened and identified in this study, which revealed the molecular mechanism of this interaction in Nsp4 induced apoptosis of host cells, and provided a valuable scientific basis for elucidating the pathogenic mechanism of PRRSV.
【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
【参考文献】
相关期刊论文 前3条
1 刘惠莉;方莹;;猪繁殖与呼吸综合征病毒非结构蛋白NSP4细胞核定位研究[J];南京农业大学学报;2011年04期
2 杨汉春,管山红,尹晓敏,甘孟侯;猪繁殖与呼吸综合征病毒的分离与初步鉴定[J];中国兽医杂志;1997年10期
3 郭宝清,陈章水,刘文兴,崔益洙;从疑似PRRS流产胎儿分离PRRSV的研究[J];中国畜禽传染病;1996年02期
，本文编号：2505948
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2505948.html
