本文選題：豬繁殖與呼吸綜合征病毒 + 核衣殼蛋白　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：豬繁殖與呼吸綜合征病毒(PRRSV)是危害世界養(yǎng)豬生產(chǎn)的重要病原,并造成巨大的經(jīng)濟(jì)損失。其核衣殼蛋白(N蛋白)是病毒粒子重要的內(nèi)部結(jié)構(gòu)蛋白及免疫原性蛋白,參與病毒RNA合成及感染性病毒顆粒裝配,且N蛋白核定位(Nuclear Localization Signal, NLS)與PRRSV致病性密切相關(guān)。SUMO是一類(lèi)重要的類(lèi)泛素蛋白,廣泛參與生物體生命活動(dòng),病毒蛋白的SUMO化修飾在抑制宿主先天性免疫、參與病毒免疫逃逸及復(fù)制中發(fā)揮重要作用。本論文圍繞PRRSV N蛋白SUMO化修飾對(duì)病毒體外復(fù)制、N蛋白亞細(xì)胞定位和抑制β干擾素產(chǎn)生的影響開(kāi)展研究,旨在從蛋白翻譯后修飾的新視角為PRRSV致病機(jī)制研究提供線索,也為抗PRRSV藥物研發(fā)提供新靶點(diǎn)。為了驗(yàn)證PRRSV蛋白的SUMO化修飾,利用分析軟件SUMOSp1.0a、SUMOplot和seeSUMO進(jìn)行預(yù)測(cè),并以SUMOE2結(jié)合酶Ubc9蛋白作為“誘餌”蛋白,采用酵母回復(fù)雜交技術(shù)篩選,發(fā)現(xiàn)Ubc9與PRRSV的Nsp1β、Nsp4、Nsp9、Nsp10及N存在相互作用。進(jìn)一步利用免疫共沉淀(Co-IP)、GST pull-down和激光共聚焦技術(shù)證實(shí)Ubc9與上述蛋白存在互作。為了分析Ubc9對(duì)PRRSV體外復(fù)制和基因組RNA合成的影響,利用RNA干擾和慢病毒包裝技術(shù)分別沉默和過(guò)表達(dá)MARC-145細(xì)胞中Ubc9,結(jié)果顯示,MARC-145細(xì)胞中過(guò)表達(dá)Ubc9在24 h可顯著抑制PRRSV復(fù)制(尸0.01)及在24 h和36 h顯著抑制PRRSV基因組RNA的合成(P0.001)。最后,為了分析SUMO化抑制劑是否有利于PRRSV復(fù)制,銀杏酸(Ginkgolic acid, GA)處理MARC-145細(xì)胞4 h,數(shù)據(jù)顯示,PRRSV在12 h和24 h于GA處理后的MARC-145細(xì)胞中的復(fù)制能力顯著高于對(duì)照組。為了闡明PRRSV N蛋白SUMO化修飾的途徑,利用定點(diǎn)突變技術(shù)對(duì)N蛋白中的賴氨酸進(jìn)行組合突變,發(fā)現(xiàn)只有當(dāng)N蛋白中的賴氨酸全部突變?yōu)榫彼釙r(shí),SUMO化修飾現(xiàn)象才消失,突變其任何一點(diǎn)或多點(diǎn)賴氨酸N蛋白仍能發(fā)生SUMO化修飾,由此證明N蛋白賴氨酸對(duì)其SUMO 化修飾是冗余的。PIAS1 (Protein inhibitor of activated STAT1)作為一種 SUMO E3 連接酶,可以促進(jìn)靶蛋白的SUMO化修飾,進(jìn)而影響靶蛋白的功能,參與基因轉(zhuǎn)錄調(diào)控過(guò)程。本研究利用SUMO化修飾體外檢測(cè)試劑盒和免疫共沉淀技術(shù)對(duì)N蛋白SUMO修飾途徑進(jìn)行鑒定,驗(yàn)證PIAS1與N蛋白的相互作用以及分析PIAS1在N蛋白SUMO化修飾及PRRSV復(fù)制中的作用。研究結(jié)果表明,PIAS1能與N蛋白相互作用,兩者共定位于胞漿中;外源轉(zhuǎn)染PIAS1不能增加N蛋白SUMO化修飾水平;在MARC-145細(xì)胞中,PIAS1的表達(dá)有利于PRRSV的復(fù)制。鑒于賴氨酸對(duì)N蛋白SUMO化修飾是冗余的,本研究構(gòu)建了系列N蛋白賴氨酸突變體并拯救病毒,進(jìn)一步驗(yàn)證N蛋白中賴氨酸與其亞細(xì)胞定位、干擾素產(chǎn)生和PRRSV復(fù)制之間的相關(guān)性。基于賴氨酸在N蛋白中的分布,以pCMV-HA-N為模板,利用定點(diǎn)突變技術(shù)構(gòu)建出系列N蛋白賴氨酸突變體;同時(shí),以PRRSV高致病性毒株JXwn06的感染性cDNA克隆(pWSK-JXwn)為骨架,定點(diǎn)突變N蛋白中的賴氨酸,共拯救出6株N蛋白賴氨酸突變病毒,分別命名為RvJXwn、RvJXNK7,28,39,52R、RvJXNmutNLS1、RvJXNmutNLS2、RvJXNmutNLS1,2 和 RvJXNKR。分析突變體拯救病毒在MARC-145細(xì)胞和原代PAMs細(xì)胞上的增殖動(dòng)態(tài),發(fā)現(xiàn)當(dāng)位于N蛋白NLS1、NLS2、NLS1,2上的賴氨酸及N蛋白中的全部賴氨酸突變?yōu)榫彼岷?突變體拯救病毒的體外增殖能力顯著低于親本病毒RvJXwn。利用激光共聚焦方法觀察N蛋白賴氨酸突變體及突變體拯救病毒的亞細(xì)胞定位,結(jié)果表明,野生型N蛋白和NmutNLS2位于細(xì)胞核核仁,而其余N蛋白賴氨酸突變體則分布在細(xì)胞核除核仁外的核質(zhì)中;突變體拯救病毒N蛋白則同親本病毒一樣分布于細(xì)胞核核仁和胞漿中。利用雙熒光素試驗(yàn)分析N蛋白及其賴氨酸突變體對(duì)IFN-β和IRFs啟動(dòng)子活性的影響,證實(shí)NK7,28,39,52R和野生型N蛋白一樣可以抑制IFN-β和IRFs啟動(dòng)子活性,而其余N蛋白賴氨酸突變體對(duì)IFN-β和IRFs啟動(dòng)子活性的抑制能力明顯減弱。針對(duì)突變體拯救病毒在MARC-145細(xì)胞和原代PAMs細(xì)胞誘導(dǎo)產(chǎn)生IFN-β能力的檢測(cè)結(jié)果表明,不同突變體拯救病毒感染宿主細(xì)胞誘導(dǎo)產(chǎn)生IFN-β的能力有明顯差異,其中RvJXNmutNLS1、RvJXNmutNLS2、RvJXNmutNLS1,2 和 RvJXNKR 在感染后 24 h 和 36h誘導(dǎo)產(chǎn)生 IFN-β 水平極顯著高于親本病毒RvJXwn(P0.001 ),而RvJXNK7,28,39,52R與親本病毒差異不顯著(P0.05)。綜上,本研究驗(yàn)證了 PRRSVNsp1β、Nsp4、Nsp9、Nsp10及N蛋白與宿主細(xì)胞蛋白Ubc9存在相互作用,證實(shí)了 PRRSVN蛋白存在SUMO化修飾現(xiàn)象,并確定賴氨酸對(duì)N蛋白SUMO化修飾是冗余的。通過(guò)構(gòu)建N蛋白賴氨酸突變體及拯救其突變病毒,證實(shí)N蛋白賴氨酸突變體可影響IFN-β和IRFs啟動(dòng)子活性及改變N蛋白的亞細(xì)胞定位;除第7,28,39,52位賴氨酸外,N蛋白其余賴氨酸與病毒的體外增殖能力密切相關(guān),且與誘導(dǎo)IFN-β水平有關(guān)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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本文編號(hào)：2055551