【作者】 郑肖娟；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 微生物学， 2007， 博士

【摘要】 传染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)属双RNA病毒家族成员,它主要侵害雏鸡体液免疫中枢器官法氏囊中的B淋巴细胞前体,导致严重的免疫抑制。由于其基因组较小,编码蛋白少,常被作为dsRNA的模式病毒进行研究。目前,对IBDV各病毒蛋白的生物学功能已有一定的了解,但是从细胞分子水平分析病毒蛋白间的相互作用、病毒与宿主相互关系的研究相对较少,而这些对于IBDV复制机制和致病机理的阐明具有重要意义。本研究以IBDV感染的宿主细胞为主要研究对象,采用荧光双标法分析感染细胞内各病毒蛋白的亚细胞定位关系,采用差异蛋白质组学方法对IBDV感染细胞的蛋白代谢变化进行了探索,并选择重要的差异表达蛋白开展深入的功能研究。VP1是IBDV基因组B节段编码的一种RNA依赖的RNA聚合酶,在基因组RNA的复制和病毒粒子的组装过程中发挥重要的作用。本研究采用长距离RT-PCR方法扩增了IBDV细胞适应毒NB株基因组B节段全长cDNA,将其编码框VP1基因亚克隆到原核表达载体pET-28a(+)上,经IPTG诱导,表达了分子量约为97kDa的VP1融合蛋白,该蛋白主要以包涵体形式存在,在体外容易发生降解。以镍柱亲和纯化的重组VPl蛋白为免疫原,制备获得4株抗IBDV-VP1的特异性鼠单克隆抗体和兔多抗血清,为深入开展IBDV复制机理研究提供了有用的抗体工具。以VP1的特异性抗体为工具,分析了pEGFP-VP1和pCI-neo-VP1真核表达质粒转染细胞以及IBDV感染细胞中VP1的表达分布情况。转染细胞内单独表达VP1及EGFP-VP1融合蛋白均以弥散状分布于胞浆内；IBDV感染细胞内,感染早期VP1主要以弥散状分布于胞浆内,感染晚期VP1聚集成大小不等的颗粒状散在分布于胞浆内。这表明VP1是一种胞浆内分布蛋白,弥散和颗粒状分布的VP1发挥不同的生物学功能,其颗粒结构与病毒复制过程有关。以VPl抗体为工具,采用荧光双标法分析VP1与衣壳蛋白VP2和VP3、丝氨酸蛋白酶VP4以及非结构蛋白VP5在鸡胚成纤维细胞(chicken embryo fibroblasts, CEF)和Vero细胞两种宿主细胞中的亚细胞定位关系。结果显示,VP1、VP2、VP3和VP5在两种细胞中的表达模式基本相同,而VP4在两种细胞中有不同的表达分布特征。其中,VP2以弥散和颗粒状分布于胞浆内,VP2颗粒比VP1大,数量比VP1少,与VP1的部分颗粒有信号重叠的现象,提示该区域可能是病毒组装的场所。与VP1相似,VP3也在胞浆内形成颗粒状结构,与VP1表现为共定位的现象,这与现有文献报道的VP1和VP3形成稳定复合物参与IBDV组装过程的结论相佐证。弥散状聚集在胞膜上的VP5与胞浆分布的VP1不存在共定位关系,表明VP5和VP1在IBDV感染过程中发挥不同的生物学功能。不同于VP1、VP2、VP3和VP5,VP4在感染细胞的胞浆和核内均有分布。在不同宿主细胞核内,VP4均形成长短不等的针状结构,在胞浆内则不同,VP4以针状结构交织成网状分布在CEF的胞浆内,以团块状结构分布于Vero细胞的胞浆核周区,胞浆内VP4信号较弱的部位则是VP1颗粒聚集的区域。进一步采用荧光双标法分析VP2、VP3和VP4在IBDV感染的鸡胚成纤维细胞系DF-1中的表达定位关系。观察发现,随着病毒感染周期的不同,VP2、VP3和VP4的表达分布模式在逐渐转变。其中,胞浆分布的VP2由弥散状逐渐聚集为颗粒状和针状结构,胞浆内的VP3也由弥散状聚集为颗粒状或中空的团块状结构；胞浆和核内分布的VP4的变化趋势相同,均呈现有弥散状→点针状→短针状→长针状和颗粒状的变化动态,其中颗粒状结构只出现在胞浆内。从定位关系看,胞浆内颗粒状和针状的VP2与VP4表现为共定位,提示在感染晚期VP2和VP4存在相互作用关系,这可能与VP4对pVP2的二次剪切即VP2的加工成熟过程有关；颗粒状的VP2和VP4大多分布在中空的团块状VP3中央,提示,这些颗粒状或团块状结构所在的区域是病毒复制组装的关键部位,病毒的复制组装主要发生在“病毒工厂”的周围区域,而病毒粒子的加工成熟则发生在“病毒工厂”中央。VP4的核内分布提示,VP4除了加工剪切多聚蛋白和VP2前体蛋白外,可能还与宿主细胞发生相互作用,在IBDV的致病中起作用。为了从细胞分子水平大规模地分析病毒与宿主细胞的相互作用关系,.本研究采用二维凝胶电泳结合质谱鉴定的差异蛋白质组学研究方法,以IBDV的易感细胞CEF为感染模型,分析IBDV感染CEF后12h、48h和96h三个不同时间细胞蛋白的表达变化动态。二维凝胶的差异表达分析显示,CEF在感染IBDV后,共有102个蛋白点出现了显著的差异表达,蛋白表达变化主要发生在感染后48h和96h。采用MALDI-TOF/TOF质谱法对102个差异表达蛋白点进行质谱鉴定,结果成功鉴定81个蛋白点(对应51种细胞蛋白),包括13种上调蛋白和38种下调蛋白。这些差异蛋白分别代表IBDV感染诱导过量表达的多聚泛素、载脂蛋白A-1、27kDa热激蛋白1、肌动蛋白、微管蛋白、真核翻译起始因子4A异构体2 (EIF4A2)、酸性核糖体磷蛋白和核糖体相关蛋白,以及IBDV感染抑制表达的参与泛素介导的蛋白降解、糖代谢、中间丝、mRNA翻译和信号转导的细胞蛋白。实时RT-PCR在转录水平上验证了质谱鉴定的38个差异表达蛋白对应基因的变化情况,确证了质谱鉴定的准确性。借助Western blot分析,应用单克隆抗体在蛋白水平上进一步确认了IBDV感染细胞中Rho蛋白解离抑制因子的表达抑制和多聚泛素的诱导表达。以上结果表明,IBDV感染主要引起了细胞内细胞骨架网络、应激反应、泛素-蛋白酶体通路、大分子合成、细胞代谢和信号转导通路的变化,这些数据为进一步研究IBDV感染的机制和致病机理提供了有用的蛋白质相关基础信息。基于IBDV感染细胞的蛋白质组学分析发现,IBDV感染诱导了宿主细胞内多种细胞骨架相关蛋白的表达变化,为了进一步分析IBDV感染对宿主细胞骨架的影响,以VPl抗体为工具,采用间接免疫荧光技术定位IBDV感染细胞,同时以F-actin特异性探针FITC-phalloidin标记微丝、抗β-tubulin和vimentin抗体标记微管和中间丝,采用荧光双标技术分析IBDV感染细胞中微丝、微管和中间丝三种骨架的亚细胞结构变化。结果显示,三种细胞骨架结构在IBDV感染细胞中均发生了特征性的变化,其中,以中心体为中心呈放射状分布的微管结构在IBDV感染后期部分或完全被破坏,胞浆内致密的vimentin中间丝骨架结构在感染后也被破坏；而微丝F-actin在感染细胞中则异常聚合,表现为在核内形成针状结构,在Vero细胞胞浆内以块状结构分布在胞浆核周区,在CEF胞浆内以针状结构交织分布。IBDV感染细胞中F-actin的表达分布变化与VP4非常相似,提示两者可能存在相互关系,因此进一步采用荧光双标技术对F-actin和VP4进行亚细胞定位关系分析。结果显示,在IBDV感染的不同宿主细胞(CEF、Vero细胞和法氏囊组织细胞)的胞浆核周区和胞核内均出现了F-actin与VP4共定位现象；而在VP4真核表达载体转染细胞中,VP4仅分布在胞浆内,且在VP4分布的区域F-actin的荧光信号更强,表现为部分共定位现象。以上结果提示,IBDV感染细胞中VP4可能与F-actin发生相互作用,核内出现的针状F-actin及针状VP4与IBDV感染有关。为了进一步分析IBDV感染细胞内VP4与F-actin的相互关系,将IBDV感染细胞用Triton X-100进行处理,分别以VP4和β-actin单抗对Triton X-100可溶成分进行免疫共沉淀分析,结果发现在可溶上清中只含少量VP4,且与β-actin单体没有相互结合关系；但在TritonX-100不溶性细胞骨架成分中检测到大量VP4,提示VP4可能与不溶性的F-actin骨架结合在一起,从而以Triton X-100不溶形式存在。为了初步探讨F-actin在IBDV感染细胞中的生物学功能,用F-actin的聚合抑制药物细胞松弛素D (cytoD)作用IBDV感染的细胞,借助TCID50测定对比药物处理前后病毒的产量和细胞上清中的病毒滴度,结果显示,中低浓度的cytoD处理显著降低病毒产量,细胞上清中的病毒滴度也明显下降,表明F-actin的解聚影响了IBDV感染性病毒粒子的产生以及病毒粒子的释放过程。作为一种胞浆复制病毒,核内出现VP4和F-actin提示VP4除了加工剪切多聚蛋白和VP2前体蛋白外,还可能与宿主细胞发生相互作用发挥功能。因此,进一步对VP4和F-actin的入核机制、互作机制以及在IBDV感染过程中的生物学功能等问题进行深入探讨,将为IBDV的复制机制和致病机理研究提供重要的线索。更多还原

【Abstract】 Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a pathogenic agent that damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius (central humoral immune organ) in young chickens, and causes severe immunosuppression. IBDV has been intensively studied as the model for dsRNA virus, since its small genomes only encode several viral proteins. To date, the biological function of viral proteins had been well characterized, but the interactions between IBDV-encoded viral proteins, and the interactions between virus and host cells are still unclear, and all these questions are essential for the potential molecular mechanism of IBDV replication and pathogenesis. In this study, we analyzed the subcellular location relationship between viral proteins, and explored the protein expression profiles of IBDV-infected cells using comparative proteomic analysis, and then analyzed the function of the differentially expressed proteins during IBDV infection.VP1, the putative viral RNA-dependent RNA polymerase encoded by segment B of IBDV, has been suggested to play essential roles in the replication of viral genomic RNAs and the assembly of viral particles. In this study, the full-length cDNA of segment B of cell-adapted IBDV-NB isolate was amplified by long RT-PCR, and then VP1 gene was subcloned into the prokaryotic expression vector pET-28a(+). After induced with IPTG, the His-tagged recombinant VP1 protein (rVP1), with an approximately molecular weight of 97 kDa, was expressed mainly as inclusion body, and the protein tends to degrade in vitro. Using the purified rVP1 by nickel-column chromatography as immunogen, polyclonal antibodies and four monoclonal antibodies (mAbs) specifically recognizing IBDV-VP1 were successfully produced, these antibodies provide useful tools for further study of the molecular mechanism of IBDV replication. Using the special antibodies against VP1, we studied the subcellular location of VP1 protein in cells infected with IBDV or transfected with eukaryotic expression plamid of VP1 (pEGFP-VP1 or pCI-neo-VPl). The results showed that VP1 diffused within the cytoplasm of transfected cells, but distributed by diffusing and forming irregularly shaped particles within the cytoplasm of IBDV-infected cells. These data implied that the formation of granule-like VP1 in infected cells may be involved in IBDV infection.Dual fluorescent staining were carried out to examine the location relationship between VP1 and capsid proteins VP2/VP3, serine protease VP4 and nonstructural protein VP5 within IBDV-infected chicken embryo fibroblasts (CEFs) and Vero cells. The results showed that the expression patterns of VP1, VP2, VP3 and VP5 in IBDV-infected CEFs and Vero cells were fairly similar, but distinct signals of VP4 were observed in two kinds of cells. VP2 located in the cytoplasm by diffusing pattern and granule-like particles, the size of VP2 particles are greater than VP1, but the numbers of VP2 particels are smaller than VP1, the VP2 particles partically colocalize with VP1, suggesting that the region of partical colocalization may be the place for viral assembly. The expression and distribution of VP3 is very similar to VP1, and VP3 co-localizes precisely with VP1, these results were consistent with the previous reports that VP3 and VP1 form a stable complex to be involved in IBDV assembly. VP5 distributes throughout the plasma membrane of IBDV-infected cells, have no colocalization with cytoplasmic VP1, suggesting that VP5 and VP1 plays distinct roles during IBDV infection. Compared with VP1, VP2, VP3 and VP5, protease VP4 distributes both in the nucleus and cytoplasm of IBDV-infected cells. In IBDV-infected CEFs, VP4 forms needle-like signal both in the nucleus and cytoplasm, while forms needle-like signal in the nucleus and agglomerate-like signal within the cytoplasm of IBDV-infected Vero cells. VP1 locates in the interspace of cytoplasmic VP4.Further, the subcellular location relationships of VP2, VP3 and VP4 in IBDV-infected DF-1 chicken fibroblast cell line were determined by dual fluorescent staining. The results showed that the expression pattern of VP2, VP3 and VP4 had gradually changed. Cytoplasmic VP2 changed from diffuse to granule-like or needle-like, cytoplasmic VP3 changed from diffuse to granule-like or hollow agglomerate. Cytoplasmic and intranuclear VP4 gradually changed from diffuse, to dot-like needle, to short needle, to long needle and granule-like particles, and the granule-like particles were only found within the cytoplasm. Granule-like and needle-like VP4 was co-localized with VP2 in the cytoplasm, indicating that VP4 may interact with VP2 during late phase, and be involved in the cleaving and maturation of precursor VP2. The particles of VP2 and VP4 mainly distribute in the center of VP3, suggesting that IBDV replication occurs in the outer region of "viral factories", and assembly and maturation of IBDV particles occurs in the inter region of "viral factories". The nuclear localization of VP4 indicated that serine protease VP4 may play important roles in IBDV pathogenesis by interacting with host cells, in addition to cleave the polyprotein and VP2 precursor.The comparative proteomes of IBDV-infected and mock-infected CEFs at different time points were analyzed using two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF protein identification. The analyses of multiple 2-DE gels revealed that a total of 102 protein spots differentially expressed during IBDV infection and the majority of protein expression changes appeared at 48 h and 96 h post infection. MALDI-TOF/TOF mass spectrometry successfully identified 81 protein spots (corresponding to 51 altered cellular proteins) of the 102 differentially expressed protein spots, including 13 up-regulated proteins and 38 down-regulated proteins. These data revealed that IBDV infection induced the overexpression of polyubiquitin, apolipoprotein A-1, heat shock 27 kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and p40 ribosomal associated protein, while considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover,38 corresponding genes of the differentially expressed proteins were quantitated by real-time RT-PCR to examine the transcriptional changes between infected and uninfected CEFs. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor and the induction of polyubiquitin during IBDV infection. These data showed that IBDV infection changed the cellular cytoskeleton, stress response, ubiquitin-proteosome pathway, macromolecular biosynthesis, energy metabolism and signal transduction. This work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis.Comparative proteomic analysis of IBDV-infected CEFs showed that multiple cytoskeleton-associated proteins were up-or down-regulated during viral infection. To further investigate the effects of IBDV infection on host cytoskeletal network, the microfilament, microtubule and intermediate filament in IBDV-infected cells (which were probed with antibodies against VP1) were detected respectively using FITC-phalloidin, antibodies againstβ-tubulin and vimentin. The results showed that IBDV infection induced the structural and distribution changes of microfilaments, microtubules and imtermediate filaments in IBDV-infected cells. Among then, the radial microtubule array anchored at the centrosome was partially or totally disrupted during late phase of infection, and the vimentin network within the cytoplasm also collapsed during IBDV infection. However, the signals of F-actin were greatly enhanced in IBDV-infected cells, with needle-like F-actin in the nucleus, with needle-like structure in the perinuclear region of cytoplasm of IBDV-infected CEFs, and with agglomerate in the cytoplasm of IBDV-infected Vero cells. The similar signal patterns were observed with anti-VP4 antibody and FITC-phalloidin in IBDV-infected cells, suggesting potential interactions between VP4 and F-actin. To further assess this possibility, their subcellular location relationship was characterized using the dual fluorescent staining. The results showed that F-actin and VP4 colocalize in both the nucleus and cytoplasm of IBDV-infected CEFs, Vero and bursal cells, but VP4 distributes within the cytoplasm of VP4-transfected cells, partically colocalize with higher signal F-actin, indicating that VP4 may interact with F-actin, and that the appearance of needle-like VP4 and F-actin in the nucleus were associated with IBDV infection. To further investigate the interaction between F-actin and VP4, IBDV-infected cells were treated with Triton X-100, and the binding activity of VP4 to monomer actin was detected using antibodies against VP4 or beta-actin by immunoprecipitation assay. The results showed that a small quantity of VP4 exists as soluble forms, and has no binding reactivity to monomer actin, while large quantity of VP4 exists as insoluble forms. These results indicated that VP4 may bind to the insoluble F-actin, and exist as insoluble forms. To assess whether F-actin is involved in IBDV infection, infected cells were treated with F-actin depolymerizing drug cytochalasin D, and the effects of drug on viral production were examined by determining the TCID50 on CEFs, the results showed that the production of infective virions were greatly decreased by treating with cytochalasin D, revealing that depolymerized F-actin potentially affects IBDV replication and release. Since IBDV replication occurs in the cytoplasm, the appearance of VP4 and F-actin in the nucleus suggested that the biological function of VP4 may largely unknown. Further investigation, including the molecular mechanism of VP4 import into the nucleus, the interaction between VP4 and F-actin, and the biological function of F-actin during IBDV infection, will greatly contribute to the elucidation of IBDV replication and pathogenesis.更多还原