【作者】 方莹；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2012， 博士

【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)引起的一种以妊娠母猪繁殖障碍、仔猪的呼吸道症状和高死亡率为特征的一种新发性病毒传染病。PRRSV基因组为不分节段的单股正链RNA,可编码14种非结构蛋白和7种结构蛋白,其中Nsp2是分子量最大且最易发生变异的一个病毒编码蛋白。2006年,由PRRSV变异株引起的高致病性PRRS在我国大规模暴发,造成上百万头猪死亡,给我国养猪业带来了巨大的经济损失。与传统的PRRSV毒株相比,高致病性PRRSV毒株能够引起更强烈的炎症反应和更高的死亡率,并成为我国流行和蔓延的主要毒株。然而,目前对这种毒株感染后引起宿主细胞的信号转导机制知之甚少。针对这种新的PRRSV,本文开展了对其诱导机体产生炎症反应的信号转导机制研究,为阐明PRRSV的致病机理提供理论依据。具体研究内容包括：1.高致病性PRRSV感染激活NF-κB信号通路的研究NF-κB是一类具有多重转录调节作用的核蛋白因子,在炎症反应、免疫反应和细胞的增殖中都有着重要的作用。利用NF-κB荧光素酶报告系统检测高致病性PRRSV对NF-κB的激活情况,发现其能显著激活NF-κB并依赖于病毒的复制,且呈剂量依赖性。进一步以PRRSV感染MARC-145细胞,检测细胞内TLR信号通路的关键接头蛋白及重要信号分子的表达情况,对PRRSV激活NF-κB的分子机制进行了初步探究。结果表明,PRRSV能上调病原模式识别受体TLR2和TLR4以及信号分子TRAF6和TAK1的表达,推测PRRSV通过同时上调模式受体TLR2和TLR4进而上调信号分子TRAF6和TAK1的表达从而激活NF-κB信号通路；同时还发现PRRSV感染的MARC-145细胞内TLR3和其他的信号蛋白并无显著上调,甚至部分细胞因子在某些时间点还出现表达下调的趋势。这提示着PRRSV在感染细胞内调控信号传导途径的复杂性,其可能是通过多种调控策略调控NF-κB的上游信号通路,而最终导致NF-κB活化的。这些结果为解释临床上PRRSV引起复杂的机体免疫反应和强烈的炎症反应补充了有用的数据。2.激活NF-κB信号通路的高致病性PRRSV非结构蛋白的筛选已有研究表明,PRRSV编码的所有结构蛋白中,N蛋白能显著激活NF-κB。为了阐明PRRSV编码的非结构蛋白在其诱导NF-κB活化中的作用,利用NF-κB荧光素酶报告系统,对高致病性PRRSV所有非结构蛋白激活NF-κB的能力进行研究,发现只有Nsp2能显著激活NF-κB,而其他的非结构蛋白对NF-κB无明显激活。3.高致病性PRRSV Nsp2激活NF-κB信号通路的分子机制研究为了解析Nsp2激活NF-κB的分子机制,首先以Nsp2的真核表达质粒转染细胞,检测IκBα和NF-κB下游炎症细胞因子的表达及NF-κB p65的磷酸化和转定位情况,发现Nsp2能诱导IκBα的降解及p65的磷酸化和转核,并激活NF-κB下游炎症细胞因子。进一步利用Nsp2截短突变体的真核表达质粒,证实Nsp2激活NF-κB的作用域位于高变区的180-480位氨基酸之间。通过对Nsp2跨膜结构域的预测和跨膜区缺失突变体的荧光素酶实验,发现其C端的跨膜结构在其细胞定位以及激活NF-κB中均发挥重要作用,推测其线粒体定位可能为其激活NF-κB所必需。进一步利用RNA干扰实验和Nsp2超表达对Nsp2诱导NF-κB的上游信号通路进行了探究,结果表明,接头蛋白TLR4和信号分子TRIF、MAVS的沉默可以抑制Nsp2对NF-κB的激活；而Nsp2的超表达可以上调TLR4、TRIF以及TRAF2的表达。这些结果表明,PRRSV Nsp2可能是同时通过TLR4和RIG-1依赖的信号途径激活NF-κB。由于RIG-1信号通路中的重要接头蛋白MAVS是一个线粒体定位蛋白,而Nsp2的线粒体定位模式改变后其激活NF-κB的能力显著降低,这说明Nsp2与MAVS的共定位对Nsp2激活NF-κB具有重要的作用。4.Nsp2与宿主蛋白RAI相互作用激活NF-κB的研究由于病毒感染动物机体后与宿主的相互作用是其致病的分子基础,本研究又从蛋白相互作用的角度对Nsp2激活NF-κB的可能机制进行探究。首先运用酵母双杂交技术从猪脑cDNA文库中筛选了与PRRSV Nsp2相互作用的宿主蛋白,并选取能与p65结合从而特异性抑制NF-κB转录活性的宿主蛋白RAI作为深入研究的对象。进一步,通过哺乳动物双杂交系统、免疫共沉淀技术、荧光共定位等实验证实了Nsp2与RAI在哺乳动物细胞内的相互作用。并利用截短突变体和酵母回返实验,确定了二者相互作用的功能域分别位于Nsp2中间的高变区和RAI的C端,由于RAI与p65的互作区域同样位于C端,推测当PRRSV感染细胞后,其编码的Nsp2可能通过与RAI相互作用从而拮抗RAI对NF-κB的抑制作用,为了验证这一推测,利用NF-κB启动子的荧光素酶报告系统检测了Nsp2与RAI相互作用对NF-κB表达的影响,结果超表达RAI显著抑制NF-κB的启动子活性,而同时超表达Nsp2与RAI时不影响NF-κB的启动子活性；进一步检测Nsp2对RAI表达水平的影响,发现Nsp2对RAI的表达水平无明显影响,这表明Nsp2拮抗RAI的NF-κB抑制功能并不是通过降低其表达水平来完成的,这也进一步证实Nsp2可能是通过拮抗RAI与p65的相互作用从而激活NF-κB的。5. PRRSV经典株与变异株引起不同程度炎症反应的分子机理研究通过比较PRRSV经典毒株CH-1a与变异毒株WUH3的Nsp2激活NF-κB的能力,发现WUH3株的Nsp2激活NF-κB的能力显著高于CH-1a株的Nsp2。由于变异毒株与经典毒株Nsp2的主要差异是中间高变区内存在30个氨基酸的不连续缺失,为了探究30个氨基酸的缺失是否是造成二者激活NF-κB能力差异显著的原因,构建了一系列Nsp2的插入和缺失突变体,并通过荧光素酶报告系统检测其激活NF-κB的能力。结果显示,1个或29个氨基酸的缺失并不是造成二者激活NF-κB能力差异的直接原因。进一步,通过荧光素酶报告系统对两种毒株的Nsp2及其突变体激活IL-6、IL-8、RANTES等炎症细胞因子的能力进行了比较,发现两种毒株的Nsp2激活IL-6、IL-8、RANTES的能力均存在显著差异,变异株的Nsp2的激活能力显著高于经典株的Nsp2,而1个或29个氨基酸的缺失并不是造成此种差异的直接原因。由于核转录因子NF-κB与炎性细胞因子IL-6、IL-8、RANTES都是参与机体炎症反应的重要细胞因子,因此以上结果为解释临床上不同毒株引起不同程度的炎症反应奠定了一定的理论基础。更多还原

【Abstract】 Porcine reproductive respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a novel infectious disease of swine. It is characterized by severe reproductive failure in sows, respiratory distress high mortality in piglets and growing pigs. The genome of PRRSV is single-stranded positive sense RNA that encodes14kinds of nonstructural proteins and7kinds of structural proteins. Among all these proteins, Nsp2is the largest and the most variable protein encoded by PRRSV.The atypical PRRS, which was caused by highly pathogenic PRRSV (HP-PRRSV), occurred in China in2006and caused tremendous economic losses due to the death of millions of pigs. Since then, the HP-PRRSV strain, which caused a more severe pneumonia and higher mortality than the classical PRRSV, has become the dominant strain in China. However, the signal transduction mechanisms involved in HP-PRRSV infection remain poorly understood. In view of the new PRRS, we analysed the mechanisms of the inflammatory responses caused by the virus in the point of signal pathway, which provide a theoretical basis for understanding molecular mechanism of PRRSV pathogenicity. The main research works were as following:1. Studies on the activation of NF-κB signal pathway induced by HP-PRRSVNF-κB is an inducible transcription factor that plays a key role in inflammation and immune responses, as well as in the regulation of cell proliferation and survival. In our study, we investigated the activation of NF-κB induced by HP-PRRSV using a luciferase reporter assay. The result showed that PRRSV activates NF-κB dependent on its replication and in a dose-dependent manner. In addition, we detect the expression pattern of various receptors and signaling molecules in MARC-145cells at diferernt time point upon PRRSV infection to evaluate the mechanisms. It was showed that the upregulated expression of receptors TLR2, TLR4and the adaptors TRAF6, TAK1was observed during PRRSV infection, which indicated that PRRSV activates NF-κB signal pathway through upregulating the expression of the TLR2and TLR4and subsequently TRAF6and TAK1signal molecules. But we also found that, the expression of other adaptors were not increased. Instead, some of them even dicreased at some certain time points, suggesting the complexity of PRRSV-induced the NF-κB activation. In this way, it was concluded that PRRSV modulates NF-κB signal pathway using a variety of different strategies, which ultimately results in the activation of NF-κB.2. Screen the specific HP-PRRSV Nsp(s) involved in NF-κB activationThe nucleocapsid (N) protein was identified as an NF-κB activator among the structural proteins encoded by PRRSV; however, it remains unclear whether the nonstructural proteins (Nsps) of PRRSV contribute to NF-κB activation. To further evaluate the mechanisms of NF-κB activation, we identified the specific PRRSV Nsp(s) involved in NF-κB activation. By screening the individual Nsps of PRRSV strain WUH3using a luciferase reporter assay, Nsp2exhibited great potential to activate NF-κB in MARC-145and HeLa cells, while this activation was not observed in cells overexpressing other viral nonstructural proteins.3. Studies on the activation of NF-κB signal pathway induced by PRRSV Nsp2In this study, overexpression of Nsp2induced IκBα degradation and nuclear translocation of NF-κB. Furthermore, Nsp2also induced NF-KB-dependent inflammatory factors, including interleukin (IL)-6, IL-8, COX-2, and RANTES. Using a series of truncated mutants of Nsp2, the functional domain responsible for NF-κB activation was located in the180-480amino acid in the HV region of Nsp2. What’s more, the predicted transmembrane region in the C terminal of Nsp2play an important part in the subcellular localization of Nsp2as well as the NF-κB activation induced by Nsp2, implied that the mitochondria location of Nsp2might be necessary for the NF-κB activation. The detailed mechanism requires further investigation. Additionally, we investigated the upstream of NF-κB signal pathway activated by Nsp2. It was identified that the TLR4adaptors and TRIF and MAVS molecules was indispensable in the NF-κB activation induced by Nsp2through conducting an SiRNA technique. Moreover, the expression pattern of several adaptors were detected in HeLa cells which an increasing amount of Nsp2was transfected. It was showed that the expression of TLR4receptors and adaptors TRIF, TRAF2were upregulated upon Nsp2overexpression. All these results suggested that both the TLR4signal pathway and the RIG-I signal pathway participated in the Nsp2-induced NF-κB activation. The mitochondria localization protein MAVS was proved to play important parts in the Nsp2-induced NF-κB activation in our study, which correlated well with the previous deduction that the mitochondria location of Nsp2was necessary for its ability of NF-κB activation. Taken as a whole, these results revealed that PRRSV Nsp2and PRRSV itself activate NF-κB signal pathway in their own differents ways, which also implied the complexity of host immune response during PRRSV infection.4. The interaction of Nsp2with RAI contributes to NF-κB activationThe protein-protein interaction of between virus and host is the molecular basis of virus pathogenicity during its infection. In present study, we explored the possible mechanism of Nsp2induced NF-κB activation by searching the functional protein-protein interaction. Host proteins that interact with PRRSV Nsp2protein were screened from pig brain cDNA library through yeast two-hybrid technology. RAI was selected for further investigation due to its biological role in inhibiting the transcription activity of p65subunit of NF-κB. Then the mammalian two-hybrid assay, the co-immunoprecipitation and the fluorescent co-localization test were performed to verify the interaction of Nsp2and RAI. Using the truncations of Nsp2and RAI, we also identified that the region of interaction located in the middle HV region of Nsp2and the C-terminal of RAI. Further biological function of the interaction was explored using a luciferase reporter assay. The result showed that Nsp2could antagonize the function of RAI in the NF-κB inhibiton probablely by antagonizing its interaction with p65. What’s more, Nsp2didn’t inhibit the expression of RAI in a western blot assay, which also implied that Nsp2might bind to RAI in competition with p65subunit to antagonize its inhbition on NF-κB transcription.5. Study on the molecular mechanism of different degree of inflammatory response between the classical PRRSV and highly pathogenic PRRSVCompared with the Nsp2of the classical PRRSV strain, the Nsp2of highly pathogenic PRRSV (HP-PRRSV) strains that possess a30amino acid (aa) discontinuous deletion in Nsp2coding region displayed greater NF-κB activation. However, the30-aa deletion was demonstrated to not be associated with NF-κB activation in a luciferase reporter assay using a series of insertion and deletion mutants. Furthermore, a promoter luciferase reporter assay was conducted to determine the ability of different Nsp2and their mutants in the activation NF-KB-regulated target gene expression, including IL-6, IL-8, and RANTES. The results showed that Nsp2and mutants from HP-PRRSV can induce higher expression level of this inflammation factors compared to that of from classic PRRSV isolate. At tha same time, the30aa deletion in Nsp2of HP-PRRSV was not related to its different levels of viral pathogenesis. Taken together, these data indicate that PRRSV Nsp2is a multifunctional protein participating in the modulation of host inflammatory response, which suggests an important role of Nsp2in pathogenesis and disease outcomes.更多还原