本文關(guān)鍵詞：豬繁殖與呼吸綜合征病毒核衣殼蛋白與細胞蛋白PARP-1、DHX9和病毒RdRp的相互作用及其對病毒增殖的影響　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

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【摘要】：豬繁殖與呼吸綜合征(PRRS)是由豬繁殖與呼吸綜合征病毒(PRRSV)引起的一種嚴重危害全球養(yǎng)豬業(yè)的傳染病。PRRSV的核衣殼(N)蛋白是表達豐度最高的病毒蛋白,與病毒基因組RNA組裝形成核衣殼,同時能夠影響多種細胞蛋白的功能。分析PRRSV感染的宿主差異表達蛋白有助于理解病毒感染導(dǎo)致的機體代謝變化及病毒的致病機制,分析N蛋白與細胞蛋白之間的相互作用有助于理解PRRSV感染過程中N蛋白發(fā)揮的作用。本論文重點探究了PRRSV N蛋白與細胞蛋白DHX9之間以及N蛋白與病毒RdRp之間的相互作用,并探討了細胞PARP-1、DHX9及病毒Rd Rp與N蛋白的相互作用對病毒增殖的影響。論文的主要研究內(nèi)容及結(jié)果如下:1.采用定量質(zhì)譜技術(shù)鑒定PRRSV感染的豬肺泡巨噬細胞(PAMs)與未感染的健康細胞得到39個差異表達蛋白。生物信息學(xué)分析的結(jié)果顯示,PRRSV感染后宿主的IL1β和IL8表達上調(diào)可能是導(dǎo)致細胞出現(xiàn)病理性變化的重要因素,而且病毒可借助不同細胞蛋白的上調(diào)或下調(diào)表達來逃避宿主的天然免疫。進一步分析通過定量質(zhì)譜鑒定得到的PRRSV N蛋白互作組的108個細胞蛋白,發(fā)現(xiàn)N蛋白與細胞的蛋白翻譯系統(tǒng)和RNA轉(zhuǎn)錄加工系統(tǒng)有密切聯(lián)系。2.通過小分子抑制劑3-AB抑制與N蛋白互作的細胞蛋白PARP-1的功能,證實PARP-1對病毒的滴度、感染率、RNA及蛋白合成過程有重要影響。經(jīng)藥物敏感性測試發(fā)現(xiàn),PRRSV在抑制劑的選擇壓力下連續(xù)傳代15次,未產(chǎn)生抗3-AB的突變病毒。這些結(jié)果表明,分析利用N蛋白的細胞蛋白互作組是鑒定影響病毒增殖的關(guān)鍵細胞蛋白的一條有效途徑。3.通過免疫共沉淀(coIP)技術(shù)和熒光顯微鏡觀察證實了PRRSV N蛋白與細胞RNA解旋酶DHX9的相互作用。通過免疫熒光技術(shù)觀察到過量表達N蛋白及PRRSV感染的細胞中內(nèi)源性DHX9都由細胞核重新定位到細胞質(zhì)中。通過干擾DHX9基因的表達發(fā)現(xiàn)病毒基因組RNA(gRNA)和長鏈亞基因組RNA(sgRNA)的合成受到抑制;而過量表達DHX9蛋白則促進gRNA和長鏈sgRNA的合成。表明細胞蛋白DHX9受病毒N蛋白招募由細胞核遷移到細胞質(zhì)中以促進病毒長鏈RNA的合成。4.我們發(fā)現(xiàn)并在不同PRRSV毒株中證實N蛋白能夠與病毒非結(jié)構(gòu)蛋白9(NSP9)發(fā)生相互作用。NSP9是病毒編碼的RNA依賴的RNA聚合酶(Rd Rp)。我們鑒定出PRRSV NSP9與N蛋白結(jié)合的結(jié)構(gòu)域位于其C末端599-646位氨基酸區(qū)域內(nèi),并且通過定點突變確定了NSP9上的E646、E608和E611以及N蛋白上的Q85是兩者結(jié)合的關(guān)鍵氨基酸。通過在感染的細胞內(nèi)過量表達與N蛋白結(jié)合的NSP9截短片段來競爭性抑制蛋白間的互作,發(fā)現(xiàn)病毒的總RNA及gRNA合成都受到抑制;表達關(guān)鍵氨基酸突變的NSP9片段的抑制效果減弱。表明N蛋白與NSP9的相互作用可能參與了病毒RNA復(fù)制轉(zhuǎn)錄的調(diào)節(jié)過程。綜上所述,我們通過高通量蛋白組學(xué)的方法分析了與PRRSV致病相關(guān)的宿主細胞蛋白;發(fā)現(xiàn)PRRSV N蛋白主要與RNA加工及蛋白翻譯過程相關(guān)的細胞蛋白相互作用;發(fā)現(xiàn)PAPR-1對PRRSV的生物學(xué)過程有重要影響;同時證實細胞蛋白DHX9與病毒N蛋白相互作用能夠促進病毒增殖。值得注意的是,我們發(fā)現(xiàn)PRRSV N蛋白能夠與病毒的RdRp相互作用并促進病毒RNA的合成。本論文為深入研究PRRSV感染過程中N蛋白與宿主細胞蛋白相互作用的生物學(xué)意義及闡明病毒負鏈RNA合成的分子機制提供了依據(jù)。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease that is caused by the porcine reproductive and respiratory syndrome virus (PRRSV) and is a serious harm to the global pig raising industry. PRRSV Nucleocapsid (N) protein expression is the highest abundance of viral proteins, assembly and viral genomic RNA form nucleocapsid, and can influence a variety of cellular protein function. Analyzing the host differentially expressed proteins of PRRSV infection is helpful to understand the changes of body metabolism and the pathogenic mechanism of virus, and to analyze the interaction between N protein and cellular proteins is helpful to understand the role of N protein in the process of PRRSV infection. This paper focuses on the interaction between PRRSV N protein and cell protein DHX9, as well as N protein and virus RdRp, and discusses the interaction between PARP-1, DHX9 and virus Rd Rp and N protein on the proliferation of virus. The main research contents and results are as follows: 1., 39 differential expression proteins of PRRSV infected porcine alveolar macrophages (PAMs) and uninfected healthy cells were identified by quantitative mass spectrometry. Bioinformatics analysis showed that the up regulation of IL1 beta and IL8 expression in host cells after PRRSV infection may be an important factor leading to pathological changes of cells. Moreover, viruses can evade the host's innate immunity by means of up regulation or down regulated expression of different cellular proteins. Further analysis of 108 cell proteins identified by quantitative mass spectrometry in the interaction group of PRRSV N protein revealed that N protein is closely related to the protein translation system of cells and RNA transcription processing system. 2., by inhibiting the function of cell protein PARP-1 interacting with N protein by small molecule inhibitor 3-AB, it is confirmed that PARP-1 has an important influence on virus titer, infection rate, RNA and protein synthesis process. The drug sensitivity test showed that PRRSV was continuously subcultured for 15 times under the selective pressure of the inhibitor, and no 3-AB resistant mutant virus was produced. These results suggest that the analysis of the cell protein interaction group using N protein is an effective way to identify the key cell proteins that affect the proliferation of the virus. 3. the interaction between PRRSV N protein and cell RNA helicase DHX9 was confirmed by immunoprecipitation (coIP) and fluorescence microscopy. By immunofluorescence, the endogenous DHX9 in the cells with excessive expression of N protein and PRRSV infection was relocated from the nucleus to the cytoplasm. By interfering with the expression of DHX9 gene, we found that the synthesis of RNA (gRNA) and long chain subgenomic RNA (sgRNA) were inhibited, while over expression of DHX9 protein promoted the synthesis of gRNA and long chain sgRNA. It is indicated that the cell protein DHX9 is recruited by the virus N protein from the cell nucleus to the cytoplasm in order to promote the synthesis of the long chain RNA. 4. we found and confirmed that N protein can interact with viral non structural protein 9 (NSP9) in different PRRSV strains. NSP9 is a RNA dependent RNA polymerase (Rd Rp) encoded by the virus. We identified that the domain of PRRSV NSP9 binding to N protein is located in the 599-646 amino acid region of C terminal, and determined the E646, E608 and E611 on NSP9, and Q85 on N protein is the key amino acid combination through site directed mutagenesis. Through over expression of NSP9 truncated fragments combined with N protein in the infected cells to compete for inhibition of protein interaction, it was found that the total RNA and gRNA of the virus were inhibited by Chengdu, and the inhibitory effect of the NSP9 fragment expressing the key amino acid mutation was weakened. The interaction of N protein and NSP9 may be involved in the regulation of RNA replication and transcription. In summary, we through high-throughput proteomic analysis of host cell proteins associated with the pathogenesis of PRRSV; found PRRSV N protein with RNA protein processing and cell protein related to the translation process of interaction; found the biological process in PAPR-1 of PRRSV have important influence; also confirmed that the cell protein DHX9 and virus N protein interaction can promote the proliferation of virus. It is noteworthy that we found that PRRSV N protein can interact with the RdRp of the virus and promote the synthesis of virus RNA. This paper provides a basis for further studying the biological significance of the interaction between N protein and host cell protein in PRRSV infection, and elucidating the molecular mechanism of viral negative RNA synthesis.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.651
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本文編號：1341317