【作者】 谢礼；

【导师】 周雪平； 洪健；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 病毒不但严重威胁人类健康和生命，还引起农作物及禽、畜和经济动物的流行性病害，造成严重的经济损失。对于病毒的研究一直是结构生物学和病理学研究的重要课题。运用传统电子显微学技术和现代结构生物学技术研究病毒蛋白聚合物的三维结构对了解这些蛋白如何发挥生物学功能，病毒如何完成生命周期具有重要意义。本文运用冷冻电镜单颗粒技术研究了麝香石竹斑驳病毒(Carnation mottle virus，CarMV)和家蚕传染性软化病病毒(Infectious flacherie virus，IFV)的三维空间结构；运用常规超薄切片电镜技术和电子断层成像技术研究了高粱花叶病毒(Sorghum mosaic virus，SrMV)柱状内含体(cylindrical inclusion，CI)的结构以及与胞间连丝的相互联系。得到以下研究结果：运用I-ELISA检测、核酸测序、SDS-PAGE蛋白电泳以及免疫吸附电镜等多种技术鉴定了CarMV云南分离物。细胞病理学观察和免疫金标记定位发现CarMV散布在寄主细胞的细胞质和液泡中，不形成内含体，没有明显的细胞病变特征，叶绿体和线粒体发生病变式膨胀，外壳蛋白在叶绿体中有分布。比较了PEG沉淀法、氯化铯密度梯度离心法和超滤法对该病毒精提纯的效果，探索了适用于冷冻电镜特殊要求的CarMV提纯方法和条件，通过氯化铯密度梯度离心法得到了适用于冻电镜三维重构研究的CarMV精提纯样品。运用冷冻电镜结合单颗粒技术研究了pH7.0的CarMV粒子，得到了分辨率为18(?)的衣壳三维立体结构，经eotest检验并以FSC的数据做图观察其曲线有收敛趋势，证明了重构的可靠性。CarMV病毒衣壳直径为354(?)，3次对称轴周围有贯穿衣壳的孔洞，亚基(subunit)排列松散。将该结果已报道的CarMV晶体结构进行比较，结果发现二者在剖分数(T值)、衣壳表面突起的数量以及衣壳的层数上存在差异。冷冻电镜结果显示水溶液状态下CarMV的T=1，表面突起为60个，为双层衣壳；而晶体学结果报道晶体状态下CarMV的T=3，表面突起为90个，为单层衣壳。在对前人报道的番茄丛矮病毒科(Tombusviridae)和南方菜豆花叶病毒属(Sobemovirus)成员晶体学数据进行比较研究中，运用‘PROSITE’软件分析了植物病毒二十面体衣壳蛋白S结构域特征肽，结合对该特征肽的拓扑学结构所进行的研究分析，发现了一些共同特征：保守序列排列方式不是连续而是间隔的；间隔距离和保守性位点的位置严格保守；位点内保守性较宽松，氨基酸性质相同即可。分离鉴定并提纯了IFV。运用冷冻电镜结合单颗粒技术研究了IFV粒子，得到分辨率为17(?)的衣壳三维立体结构，经eotest检验并以FSC的数据做图观察其曲线有收敛趋势，证明了重构的可靠性。重构结果显示IFV粒子遵循二十面体P=3对称规则，直径为302.4(?)，衣壳厚度为15(?)。三维密度图显示IFV衣壳粒子表面光滑，无毛刺和突起。将IFV衣壳三维结构与亲缘关系较接近的蟋蟀麻痹病毒(CrPV)(昆虫类小RNA病毒)和人类鼻病毒(HRV14)、门戈病毒(Mengovirus)、脊髓灰质炎病毒(Poliovirus)和口蹄疫病毒(FMDV)(脊椎动物小RNA病毒)的衣壳三维结构作比较，发现IFV衣壳的三维结构更接近CrPV。运用‘phyre’肽链折叠预测工具得到了IFV-VP2、IFV-VP3的肽链折叠结构，并将预测结果同CrPV晶体结构的肽链进行比较，发现相似度较高。从IFV粒子光滑的表面缺乏明显的沟壑来看，IFV逃避寄主追踪并不是采用类似“峡谷”假说的机制，而是另一种目前未知的机制。运用常规超薄切片电镜观察结合ET三维重构技术研究了SrMV侵染的玉米叶片细胞中柱状内含体(CI)超微结构以及与胞间连丝之间的相互联系。结果显示：细胞质中分布了大量柱状内含体，其基部与粗面内质网或细胞膜相连，线状病毒粒子纵向排列在柱状体的蛋白板上；在侵染早期CI垂直于细胞壁，基部通过细胞膜与胞间连丝相连，每个CI的中央轴芯和辐射状排列的蛋白板可与多个胞间连丝通道相连，胞间连丝直径明显增大，并有线状病毒样粒子通过CI与胞间连丝连接通道穿越的现象；CI与胞间连丝的连接也存在于维管束组织中，游离的CI存在于筛管内。对厚切片(200nm)进行ET三维重构得到的三维立体模型证实了CI的结构以及与胞间连丝的连接关系。通过对该模型进行分割、渲染，结合超薄切片电镜观察所得到的结果进行CI生物学功能分析，我们初步认为：(1) Potyvirus的柱状内含体在病毒胞间运动中起着重要辅助作用，CI能定向连接胞间连丝，帮助纵向定位在CI蛋白板上的病毒粒子完成胞间运动。(2) CI中央的孔道并不是由单独的结构部件所组成，单个CI可以同时连接几个胞间连丝通道，输送多个病毒粒子；一旦胞间运动完成后则脱离细胞壁，游离于细胞质中。(3)筛管与伴胞及其他细胞间的转运也是依靠CI辅助，病毒的长距离运输可能是以完整粒子形式进行的。更多还原

【Abstract】 Viruses not only do harm to human health, but also infect crops and breeding animals,causing severe economic losing. Structure studies of viruses are important subjects instructural biology and cell pathology. Using traditional experimental methods of electronmicroscopy (EM) combined with structural biological methods to study three dimensionalstructure of virus protein polymers can help us known how these protein polymers exertbiological function, and how viruses complete their life cycle. We report the structuralstudies of virus protein polymers by using cryo-electron microscopy and three dimensionalreconstruction to study the capsids structure of Carnation mottle virus (CarMV) andInfectious flacherie virus (IFV); traditional ultra thin section and electron tomography wereused to study the cylindrical inclusion (CI) structure of Sorghum mosaic virus (SrMV) andthe relationship between CI and plasmodesma (PD).Using multiple methods such as I-ELISA?, nucleic acid sequencing, SDS-PAGE andimmunolabelling in EM, we identified CarMV Yunnan isolation. CarMV particles werefound to be districbuted in cytoplasm and vacuole in EM. CarMV have no cell inclusion incytoplasm and no obvious cytopathological change can be found. The chloroplast andmitochondria of host cell badly swell after CarMV infection. The capsid proteins of CarMVdistributing in chloroplast. By comparing the purification effect of three subtle purificationmethods such as PEG deposition, CsCl density gradient centrifugation and centrifugal filter,we find that CsCl density gradient centrifugation can purified virions suitable for cryoelectron microscopy study. Using cryo-electron microscopy combined with single particlereconstruction method, we obtained the resolution of 18(?) for the capsid structure of CarMVin pH7.0 solution.. After eotest calculation and checking the convergence of FSC curve, weconfirm that the reconstruction is reliable. The diameter of CarMV capsid is 354(?). Thereare shell perforative chanels near 3-fold axis. The arrangement of subunits is incompact. Bycomparing this structural result with the reported crystal structure, we find differences on Tnumber, number of spikes in the surface of the shell and numbers of capsid shell, The cryoEM result of CarMV in solution show T=1, there are 60 spikes in the surface and there aretwo layers of shell. The structure of crystalloid CarMV show T=3, there are 90 spikes in thesurface and there is only one layer of shell. The crystal structures of Tombusviridae andSobemovirus were cpmpared using program ’PROSITE’ combined with structural analysis,we find that the ’plant virus icosahedral capsid protein S region signature’ motif hascommon chracteristics: the array of conservative sequences is not successive but distant; thedistances between key sites are strict conservative; the amino acids within key sites are notstrict conservative.We purified IFV particles. Using cryo-electron microscopy combined with single particlereconstruction method, the capsid structure of IFV capsid was studied and the resolution of17(?) was obtained. After eotest calculation and checking the convergence of FSC curve, weconfirm that the reconstruction is reliable. The diameter of IFV capsid is 302.4(?), thethickness of shell is 15(?). Three dimensional density map show the subunits of IFV followthe P=3 icosahedral symmetry folding pattern. The capsid shell surface of IFV is smoothwithout spikes or protrudes. By comparing the IFV structures with insect picorna (-like) virus CrPV and picornavirus HRV14, Mengovirus, poliovirus, FMDV, we find that the IFVresembles to CrPV in lacking of ’Rossmann canyon’, indicating that the mechanism of IFVescaping from host immunity system tracing is different from the mechanism discovered inHRV14 reported as ’Rossmann canyon’. Using program ’phyre’, we predict the polypeptidefolding pattern of IFV-VP2 and IFV-VP3 subunit. The polypeptide topology of these twoIFV subunits and the their location at the capsid surface resemble to CrPV’s.The CI structure of SrMV and the relationship between CI and plasmodesma (PD) inSrMV infected maize cell were studied by using traditional ultra thin section and electrontomography. The following results were obtained: many CIs distribute in cytoplasm, thebase part of CI conjunct with ER or cell membrane; linear virus particles disposedlengthwise on the plates of CI; the CI in early infecting stage connect with cell wallvertically, the base of CI connect with PD; there are multiple passageways of virus betweenCI and PD, not only through the central channel of CI; the diameter of PD is enlarged; theCI bodies and the connection of CI and PD are also exist in vascular bundle cells. Threedimensional model of CI obtained by ET reconstruction method also confirmed therelationship between CI and PD. After segmentation and rending to the model, thebiological function of CI were predicted: (a) The cylindrical inclusion of potyviruses playsan important role in virus transportation between cells. CI can help virus lengthwise locatingin its plate and then pass through PD. (b) The central channel of CI is not composed ofindependent configurable components. One single CI can connect with multiple PD tocompose multiple channels to transport viruses. Once the virus transportation finished, CIdisengage cell wall and dissociate in cytoplasm. (c) The virus transportation in companioncell and sieve tube cell is depend on CI. Long distance transportation of virus is by thepattern of intact virus particles.更多还原