【作者】 王丽；

【导师】 于康震； 杨汉春；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 猪繁殖与呼吸综合征病毒(PRRSV)是危害全球养猪业的重要病原之一。PRRSV的非结构蛋白2(Nsp2)是其基因组编码的分子量最大的蛋白,参与病毒感染过程的多个环节并发挥重要功能。本研究通过全面筛选与Nsp2相互作用的宿主细胞蛋白以及绘制Nsp2与宿主细胞蛋白的互作图谱,进一步对Nsp2与宿主细胞蛋白Bcl2相关抗凋亡基因6(BAG6)和凋亡诱导因子1(AIF1)的相互作用及其影响PRRSV复制的分子机制进行了研究,以期为揭示Nsp2在PRRSV感染过程中的生物学功能提供科学依据。利用PRRSV JXwn06的感染性cDNA克隆,成功构建并拯救出Nsp2区域融合表达外源标签3xMyc的重组病毒(命名为RvMyc-JXwn)。以重组病毒感染MARC-145细胞,采用免疫沉淀结合串联质谱技术,共筛选出617个与Nsp2相互作用的宿主细胞蛋白,其中高度可信(P<0.01)的蛋白共285个。利用生物信息学技术,对285个宿主细胞蛋白进行了功能分析。结果表明,与Nsp2互作的宿主细胞蛋白主要参与病原致病、蛋白翻译、免疫系统、神经系统以及信号转导等相关的细胞通路。基于已知的Nsp2功能和相关宿主细胞蛋白功能,对Nsp2与宿主细胞蛋白BAG6和AIFl的互作及其分子机制进行了研究。利用免疫共沉淀(Co-IP)技术验证了Nsp2与BAG6的相互作用,并确定了相互作用的区域为Nsp2N端的半胱氨酸蛋白酶结构域和BAG6N端的泛素样结构域,而Nsp2的半胱氨酸蛋白酶活性位点与二者的相互作用无关。激光共聚焦分析显示,PRRSV感染和利用Nsp2真核表达质粒转染细胞均能诱导BAG6从细胞核转移至细胞质,并与Nsp2共定位。经体内、外泛素化降解试验分析表明,Nsp2的去泛素化酶活性可以抑制其自身被BAG6招募的泛素-蛋白酶体系统降解；免疫电镜分析显示,BAG6有助于Nsp2定位于内质网来源的双层膜泡结构。进一步发现PRRSV感染和Nsp2表达均能导致caspase12的激活,诱导细胞产生由内质网应激所介导的细胞凋亡,表明凋亡的发生可能与Nsp2通过自身去泛素化酶活性,抑制BAG6招募的泛素-蛋白酶体系统对细胞中缺陷蛋白的降解,从而导致细胞内蛋白泛素化水平降低和错误蛋白的积累有关。此外,证实BAG6的表达有利于PRRSV的复制,但对病毒释放没有明显的影响。利用Co-IP技术证实了Nsp2和AIFl之间的相互作用,并确定了Nsp2与AIFl相互作用的区域为N端的半胱氨酸蛋白酶结构域,且Nsp2的酶活性位点对其相互作用起着决定性作用。激光共聚焦分析显示,在PRRSV感染和利用Nsp2真核表达质粒单独转染细胞的情况下,Nsp2均能与AIF1在细胞质中共定位,并且能够诱导AIF1进入细胞核。泛素化降解试验结果表明,PRRSV感染和Nsp2表达均能阻止AIF1被泛素-蛋白酶体系统降解,Nsp2的去泛素化酶活性在该过程中发挥重要作用。进一步发现PRRSV感染和Nsp2表达均能诱导AIF1介导的非caspase依赖性细胞凋亡的发生,AIF1的表达有利于病毒感染过程中细胞内ATP的产生,并且可以促进病毒的复制。综上所述,我们的研究筛选出了可与PRRSVNsp2互作的宿主细胞蛋白,绘制出Nsp2与宿主细胞蛋白的互作图谱,为研究Nsp2与宿主细胞的相互作用提供了重要线索和依据；揭示了Nsp2与宿主细胞蛋白BAG6和AIF1互作的分子机制及其对PRRSV复制的影响,为进一步探索Nsp2在PRRSV复制和致病机制中的生物学功能及其分子机制提供了新的思路和方向。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important etiological agents affecting swine industry worldwide. The nonstructural protein2(Nsp2) of PRRSV is the largest viral protein that plays important roles in the PRRSV infection. In the present study, the host cellular proteins that interact with the Nsp2of PRRSV were first identified in the MARC-145cells and an interactome profile between them was drawn; Next, the molecular mechanisms of the interaction of Nsp2with the host cellular proteins, BCL2-associated athanogene6(BAG6) and apoptosis-inducing factor1(AIF1), and the roles of their interactions in the PRRSV replication were explored. The objective of this study is to provide scientific evidences for elucidating the biological fuctions of Nsp2during the PRRSV infection.The infectious cDNA clone of JXwn06was used to generate a chimeric cDNA clone that contains3×Myc tag insertion in its Nsp2-coding region. A recombinant virus was rescued from the chimeric cDNA clone and named RvMyc-JXwn. The host cellular proteins that interact with the Nsp2of PRRSV were immunoprecipitated from the MARC-145cells infected with RvMyc-JXwn with an anti-Myc antibody, and617cellular proteins interacting with Nsp2were then identified by LC-MS/MS. Out of these proteins,285proteins with high Confidence Icons (P<0.01) were used for bioinformatics analysis. Functional analysis of the interactome profile indicated that these proteins were involved in the cellular pathways associated with infectious disease, translation, immune system, nervous system and signal transduction.Given to the fuctions of Nsp2and cellular proteins, we explored the molecular mechanisms of the interaction between Nsp2and cellular proteins-BAG6and AIF1. The binding regions of BAG6and Nsp2were identified by Co-IP. Our results showed that the N-terminus but not the cysteine protease (CP) active sites of Nsp2interacted with the N-terminal ubiquitin-like (UBL) domain of BAG6. The confocal microscopy analysis showed that BAG6localized in the nucleus of the cells re-localized to the cytoplasm and co-localized with Nsp2following PRRSV infection in MARC-145cells or PAMs or Nsp2-expressing plasmid transfection in MARC-145cells. Subsequently, we demonstrated that BAG6contributed to target Nsp2to the endoplasmic reticulum (ER)-originated double-membrane vesicles (DMVs). Additionally, the ER stress-mediated apoptosis was discovered in PRRSV-infected cells, and Nsp2induced the ER stress-mediated apoptosis by deubiquitinating BAG6-bound defective proteins and preventing them from degrading, which resulted in the accumulation of defective protein during PRRSV infection. Finally, we found that BAG6played a crucial role in promoting the PRRSV replication, whereas it did not affect the virus release.The binding regions of AIF1and Nsp2were identified by Co-IP. The results showed that the N-terminus of Nsp2interacted with AIF1, whereas the CP active sites were necessary for this interaction. The confocal microscopy analysis indicated that Nsp2could co-localize with AIF1in the cytoplasm and lead to the nuclear translocation of AIF1following PRRSV infection in MARC-145cells or PAMs or Nsp2-expressing plasmid transfection in MARC-145cells. We also found that Nsp2could stabilize AIF1by preventing proteasome from degrading it. In the present study, we demonstrated for the first time that PRRSV infection and Nsp2expression could induce the caspase-independent apoptosis mediated by AIF1. In addition, our data suggested that AIF1played a key role in generating ATP during PRRSV infection and enhanced the replication of PRRSV.As a whole, we identified the host cellular proteins that interact with the Nsp2of PRRSV and drawn an interactome profile of Nsp2with host cellular proteins, providing important clues and evidence for exploring the interaction between Nsp2and cellular proteins. We revealed the molecular mechanisms affecting viral replication by the interaction of PRRSV Nsp2with BAG6or AIF, providing an insight and novel way for further exploration of the biological functions and molecular mechanisms of Nsp2which is involved in the replication and pathogenesis of PRRSV.更多还原