豬繁殖與呼吸綜合征病毒Nsp4與細(xì)胞色素C1相互作用及其誘導(dǎo)細(xì)胞凋亡的分子機(jī)制
發(fā)布時(shí)間：2019-06-25 19:39
【摘要】：豬繁殖與呼吸綜合征病毒(PRRSV)是嚴(yán)重危害全球養(yǎng)豬生產(chǎn)的重要病原,高致病性PRRSV的出現(xiàn)和流行給我國養(yǎng)豬業(yè)造成了巨大的經(jīng)濟(jì)損失。PRRSV基因組可編碼產(chǎn)生至少14個(gè)非結(jié)構(gòu)蛋白,其中非結(jié)構(gòu)蛋白4 (Nsp4)具有3C樣絲氨酸蛋白酶活性,在病毒的復(fù)制、抑制宿主天然免疫反應(yīng)和誘導(dǎo)宿主細(xì)胞凋亡的過程中具有重要作用。本研究以PRRSV非結(jié)構(gòu)蛋白Nsp4為研究對象,篩選與鑒定出與PRRSV Nsp4相互作用的豬肺泡巨噬細(xì)胞蛋白—細(xì)胞色素c1(Cytochrome c1,Cyto.c1),進(jìn)一步研究了其相互作用及其影響Nsp4誘導(dǎo)細(xì)胞凋亡的分子機(jī)制,以期揭示Nsp4新的生物學(xué)功能,并為闡明PRRSV的分子致病機(jī)制提供科學(xué)依據(jù)。利用PRRSV高致病性毒株JXwn06,采用慢病毒包裝技術(shù),成功構(gòu)建并拯救插入JXwn06 Nsp4基因及其酶活位點(diǎn)單點(diǎn)突變的重組慢病毒,并成功侵染MARC-145細(xì)胞,最終獲得表達(dá)融合蛋白的Nsp4單點(diǎn)突變體傳代細(xì)胞系。由于Nsp4可誘導(dǎo)細(xì)胞凋亡,未能獲得Nsp4穩(wěn)定表達(dá)的細(xì)胞系。為研究Nsp4誘導(dǎo)細(xì)胞凋亡的分子機(jī)制,以Nsp4作為“誘餌”蛋白,利用豬肺泡巨噬細(xì)胞cDNA文庫和酵母雙雜交技術(shù),成功篩選獲得與凋亡調(diào)控和凋亡信號傳導(dǎo)相關(guān)蛋白—細(xì)胞色素c1(Cyto.c1),并通過酵母回交試驗(yàn)加以確認(rèn)。利用免疫共沉淀技術(shù)(Co-IP)和激光共聚焦試驗(yàn)驗(yàn)證了 Nsp4和Cyto.c1在宿主細(xì)胞中相互作用,并確定其相互作用的區(qū)域分別為Nsp4的N端(1～160aa)和 Cyto.c1 的 N 端(1～230aa)。利用siRNA技術(shù)沉默MARC-145細(xì)胞中內(nèi)源性Cyto.c1表達(dá),分析Nsp4與Cyto.c1相互作用對Nsp4誘導(dǎo)細(xì)胞凋亡的影響。結(jié)果表明,干擾Cyto.c1表達(dá)后,Nsp4所誘導(dǎo)的細(xì)胞凋亡被顯著抑制。同時(shí),發(fā)現(xiàn)在PRRSV感染和質(zhì)粒轉(zhuǎn)染的情況下,Nsp4可以剪切Cyto.c1,剪切能力是由其3C樣絲氨酸蛋白酶活性所決定的,并呈現(xiàn)劑量依賴性。進(jìn)一步研究發(fā)現(xiàn),Cyto.c1的E230aa～G231aa是決定Nsp4剪切活性的關(guān)鍵位點(diǎn)。Cyto.c1被剪切后形成的Cyto.c1/1～230aa蛋白可明顯激活Caspase-3,同時(shí)引起線粒體的片段化,進(jìn)而導(dǎo)致線粒體損傷,激活線粒體凋亡通路,最后誘導(dǎo)細(xì)胞凋亡的發(fā)生。綜上所述,本研究篩選并鑒定出PRRSV Nsp4可與宿主細(xì)胞蛋白Cyto.c1相互作用,揭示了這一相互作用在Nsp4誘導(dǎo)宿主細(xì)胞凋亡中的分子機(jī)制,為闡明PRRSV的致病機(jī)理提供了有價(jià)值的科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S852.65
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S852.65
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