猪繁殖与呼吸综合征病毒核衣壳蛋白与细胞蛋白PARP-1、DHX9和病毒RdRp的相互作用及其对病毒增殖的影响

发布时间：2017-12-27 10:20

  本文关键词：猪繁殖与呼吸综合征病毒核衣壳蛋白与细胞蛋白PARP-1、DHX9和病毒RdRp的相互作用及其对病毒增殖的影响　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

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【摘要】：猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种严重危害全球养猪业的传染病。PRRSV的核衣壳(N)蛋白是表达丰度最高的病毒蛋白,与病毒基因组RNA组装形成核衣壳,同时能够影响多种细胞蛋白的功能。分析PRRSV感染的宿主差异表达蛋白有助于理解病毒感染导致的机体代谢变化及病毒的致病机制,分析N蛋白与细胞蛋白之间的相互作用有助于理解PRRSV感染过程中N蛋白发挥的作用。本论文重点探究了PRRSV N蛋白与细胞蛋白DHX9之间以及N蛋白与病毒RdRp之间的相互作用,并探讨了细胞PARP-1、DHX9及病毒Rd Rp与N蛋白的相互作用对病毒增殖的影响。论文的主要研究内容及结果如下:1.采用定量质谱技术鉴定PRRSV感染的猪肺泡巨噬细胞(PAMs)与未感染的健康细胞得到39个差异表达蛋白。生物信息学分析的结果显示,PRRSV感染后宿主的IL1β和IL8表达上调可能是导致细胞出现病理性变化的重要因素,而且病毒可借助不同细胞蛋白的上调或下调表达来逃避宿主的天然免疫。进一步分析通过定量质谱鉴定得到的PRRSV N蛋白互作组的108个细胞蛋白,发现N蛋白与细胞的蛋白翻译系统和RNA转录加工系统有密切联系。2.通过小分子抑制剂3-AB抑制与N蛋白互作的细胞蛋白PARP-1的功能,证实PARP-1对病毒的滴度、感染率、RNA及蛋白合成过程有重要影响。经药物敏感性测试发现,PRRSV在抑制剂的选择压力下连续传代15次,未产生抗3-AB的突变病毒。这些结果表明,分析利用N蛋白的细胞蛋白互作组是鉴定影响病毒增殖的关键细胞蛋白的一条有效途径。3.通过免疫共沉淀(coIP)技术和荧光显微镜观察证实了PRRSV N蛋白与细胞RNA解旋酶DHX9的相互作用。通过免疫荧光技术观察到过量表达N蛋白及PRRSV感染的细胞中内源性DHX9都由细胞核重新定位到细胞质中。通过干扰DHX9基因的表达发现病毒基因组RNA(gRNA)和长链亚基因组RNA(sgRNA)的合成受到抑制;而过量表达DHX9蛋白则促进gRNA和长链sgRNA的合成。表明细胞蛋白DHX9受病毒N蛋白招募由细胞核迁移到细胞质中以促进病毒长链RNA的合成。4.我们发现并在不同PRRSV毒株中证实N蛋白能够与病毒非结构蛋白9(NSP9)发生相互作用。NSP9是病毒编码的RNA依赖的RNA聚合酶(Rd Rp)。我们鉴定出PRRSV NSP9与N蛋白结合的结构域位于其C末端599-646位氨基酸区域内,并且通过定点突变确定了NSP9上的E646、E608和E611以及N蛋白上的Q85是两者结合的关键氨基酸。通过在感染的细胞内过量表达与N蛋白结合的NSP9截短片段来竞争性抑制蛋白间的互作,发现病毒的总RNA及gRNA合成都受到抑制;表达关键氨基酸突变的NSP9片段的抑制效果减弱。表明N蛋白与NSP9的相互作用可能参与了病毒RNA复制转录的调节过程。综上所述,我们通过高通量蛋白组学的方法分析了与PRRSV致病相关的宿主细胞蛋白;发现PRRSV N蛋白主要与RNA加工及蛋白翻译过程相关的细胞蛋白相互作用;发现PAPR-1对PRRSV的生物学过程有重要影响;同时证实细胞蛋白DHX9与病毒N蛋白相互作用能够促进病毒增殖。值得注意的是,我们发现PRRSV N蛋白能够与病毒的RdRp相互作用并促进病毒RNA的合成。本论文为深入研究PRRSV感染过程中N蛋白与宿主细胞蛋白相互作用的生物学意义及阐明病毒负链RNA合成的分子机制提供了依据。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651
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本文编号：1341317