宿主细胞蛋白p21和ILF2调控猪繁殖与呼吸综合征病毒复制的分子机制

发布时间：2018-03-07 00:08

  本文选题：猪繁殖与呼吸综合征病毒　切入点：nsp11　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征病毒(PRRSV)是严重影响全球养猪业的重要病原之一。近来年,PRRSV的非结构蛋白(nsp)在其基因组转录、复制调控和拮抗机体的天然免疫应答中的作用受到广泛关注。本研究分析了 PRRSVnsp11对宿主细胞蛋白p21的降解作用及其生物学意义,探讨了病毒的nsp9和nsp2与宿主细胞的白细胞介素增强子结合因子2 (ILF2)的相互作用及其对病毒复制的调控效应,以期揭示nsp11、nsp9和nsp2在PRRSV复制过程中的作用,为理解PRRSV复制调控的分子机制提供科学依据。采用Western Blot分析了 PRRSV感染MARC-145细胞中p21蛋白的变化。结果显示,PRRSV感染呈剂量依赖性下调MARC-145细胞中p21蛋白水平。流式细胞术分析表明,PRRSV 可通过下调p21蛋白而促进细胞进入S期。分别用血清饥饿、胸苷双阻断和诺苯达唑处理,将MARC-145细胞同步化至G0/G1、S和G2/M期后接种PRRSV,测定病毒滴度。结果显示,同步化至S期的细胞有利于PRRSV复制。进一步采用CRISPR/Cas9系统证实敲除MARC-145细胞p21基因有利于病毒复制。将表达PRRSV各个非结构蛋白和结构蛋白的质粒与表达p21的质粒共同转染HEK 293FT细胞,进行Western Blot分析。结果显示,nsp11可明显下调p21蛋白。构建失活核酸内切酶活性和去泛素化酶活性的nsp11突变体,与表达p21的质粒共同转染HEK293FT细胞。结果表明,nsp11的核酸内切酶活性对其下调p21蛋白至关重要。Real-time PCR分析表明,PRRSV感染和表达nsp11的质粒转染细胞后,p21mRNA水平均无明显变化。降解试验显示nsp11通过蛋白酶体途径降解p21,泛素化分析证实nsp11通过非泛素依赖的蛋白酶体途径降解p21。以上结果表明,PRRSV利用其nsp11的核酸内切酶活性下调细胞p21蛋白,促进细胞进入S期,从而利于病毒自身复制。利用慢病毒表达系统结合免疫沉淀技术及质谱分析,筛选出52个与PRRSV nsp9相互作用的细胞蛋白,选取ILF2进行下一步研究。将表达ILF2的质粒和表达nsp9或nsp2的质粒共同转染HEK 293FT细胞,通过免疫共沉淀(Co-IP)证实外源ILF2与nsp9和nsp2均存在相互作用。进一步在过表达nsp9或PRRSVJXwn06感染的MARC-145细胞中证实内源性ILF2与nsp9或nsp2存在相互作用。利用Co-IP证实nsp9的RdRp结构域与ILF2相互作用,而nsp2的不同截短体均与ILF2相互作用。此外,PRRSV感染可使MARC-145细胞和PAMs中的部分ILF2从细胞核移位至细胞质,与nsp9和nsp2共定位。在MARC-145细胞中,利用siRNA敲低和慢病毒过表达试验证实ILF2可负调控PRRSV复制。以上结果表明,细胞蛋白ILF2与PRRSVnsp9和nsp2均存在相互作用,且这种相互作用不利于病毒自身复制。综上所述,PRRSV利用其nsp11的核酸内切酶活性通过非泛素依赖的蛋白酶体途径降解细胞内p21蛋白水平,促进细胞进入S期,从而有利于病毒复制。细胞蛋白ILF2与PRRSV的nsp9和nsp2存在相互作用,且ILF2对病毒复制具有负调控效应。以上结果有助于进一步理解PRRSV与细胞间的相互作用及nsp 11、nsp9和nsp2在PRRSV复制过程中的作用,为阐明PRRSV复制调控的分子机制提供了科学依据。
[Abstract]:Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of serious impact on the global pig industry. In recent years, non structural protein PRRSV (NSP) in its genome transcription, replication and regulation of the innate immune response in the role of antagonistic organism has attracted extensive attention. This study analyzed the degradation effect of PRRSVnsp11 on host cells protein p21 and its biological significance of interleukin Nsp9 and Nsp2 virus and host cell factor enhancer binding factor 2 (ILF2) and its interaction effects on viral replication, in order to reveal the role of nsp11, Nsp9 and Nsp2 in the PRRSV replication process, to provide a scientific basis for understanding the molecular mechanisms of PRRSV copy control. Using Western Blot to analyze the changes of p21 protein in MARC-145 cells infected with PRRSV. The results showed that PRRSV infection was dose dependent downregulation of p21 protein level in MARC-145 cells by flow cytometry. FCM analysis showed that PRRSV can downregulate p21 protein and promote cells to enter S phase respectively. Serum starvation, thymidine double block and Nobel albendazole treatment, MARC-145 cells were synchronized to G0/G1, S and G2/M after PRRSV inoculation, the virus titer was determined. The results show that the synchronization to S phase cells for PRRSV replication. Further using CRISPR/Cas9 system confirmed that knockdown of MARC-145 cells with p21 gene for virus replication. The expression of PRRSV and expression plasmid all non structural and structural proteins of p21 co transfected with plasmid HEK 293FT cells, Western Blot analysis. The results show that nsp11 can decrease p21 protein inactivation. Construction of nucleic acid enzyme activity and deubiquitinating enzyme activity of nsp11 mutants, and the expression of p21 plasmid was co transfected into HEK293FT cells. The results showed that the nsp11 endonuclease activity on the expression of p21 protein is.Real-time PCR analysis showed that PRRSV infection and expression of nsp11 plasmid transfected cells after p21mRNA levels did not change significantly. The degradation test showed that nsp11 degradation via the proteasome pathway p21, ubiquitination analysis confirmed that nsp11 through the ubiquitin dependent proteasome degradation pathway of p21. above the PRRSV using its nsp11 endonuclease activity by p21 cells protein, promote cells to enter S phase, which is conducive to viral replication. The expression system combined with immunoprecipitation technology and mass spectrometry using lentivirus, screened 52 PRRSV and Nsp9 interacting protein, ILF2 is selected for the next study. The expression and expression of ILF2 plasmid Nsp9 or Nsp2 co transfected with plasmid HEK 293FT cells by CO immunoprecipitation (Co-IP) confirmed that exogenous ILF2 with Nsp9 and Nsp2 have interaction. Further in the over expression of Nsp9 or PRRSVJXwn06 infected MARC-145 cells The interaction between endogenous ILF2 and Nsp9 or Nsp2 confirmed. By Co-IP confirmed the interaction of Nsp9 domain and ILF2 RdRp structure, and different truncated Nsp2 were interacted with ILF2. In addition, PRRSV infection can make the ILF2 and PAMs in MARC-145 cells from the nucleus translocated to the cytoplasm, CO localization with Nsp9 and Nsp2. In MARC-145 cells, using siRNA knockdown and overexpression of lentivirus test confirmed that ILF2 can negatively regulate PRRSV replication. These results indicate that cell protein ILF2 with PRRSVnsp9 and Nsp2 have interaction, and this interaction is not conducive to the virus replication. In summary, the protein level of p21 proteasome degradation pathway of PRRSV cells using nsp11 the endonuclease activity through the ubiquitin dependent, promote cells to enter S phase, which is conducive to viral replication. The interaction between ILF2 and PRRSV cell protein Nsp9 and Nsp2, and ILF2 Viral replication plays a negative regulatory role. The above results will help further understand the interaction between PRRSV and cells, as well as the role of NSP 11, Nsp9 and Nsp2 in the process of PRRSV replication, and provide a scientific basis for elucidating the molecular mechanism of PRRSV replication regulation.

【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

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