Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles.
Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles.
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DOI：
10.1016/j.virol.2007.05.038
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发表时间：
2007-10
期刊：
影响因子：
3.7
通讯作者：
Xuekui Yu;M. Qiao;I. Atanasov;Zongyi Hu;Takanobu Kato;T. Liang;Z. H. Zhou
中科院分区：
文献类型：
--
作者：
Xuekui Yu;M. Qiao;I. Atanasov;Zongyi Hu;Takanobu Kato;T. Liang;Z. H. Zhou
丙型肝炎病毒（HCV）的结构细节一直难以捉摸，因为缺乏一个强大的组织培养系统，从感染源产生足够量的病毒粒子进行深入的三维（3D）结构分析。使用负染色和冷冻电子显微镜（cryoEM），我们表明，从细胞培养物中分离的HCV病毒粒子有一个相当均匀的大小为500 μ m的直径和重组表达的HCV样颗粒（HCV-LP）具有相似的形态，生物物理和抗原特性，尽管不同大小的颗粒。在存在和不存在与E1糖蛋白结合的单克隆抗体的情况下，从具有与HCV病毒体相同大小的HCV-LP获得3D重建。HCV-LP的3D重建揭示了一个多层结构，具有以“鱼骨”配置排列的平滑外层密度。与抗E1抗体复合的颗粒的重建显示E1表位的位点被暴露并且围绕5-、3-和2-倍轴。抗E1抗体的结合模式和登革病毒E糖蛋白的结构拟合到我们的3D重建进一步表明，HCV-LP E1和E2蛋白形成四聚体（或异源二聚体的二聚体），其在形态和功能上对应于黄病毒E同源二聚体。HCV颗粒的首次3D结构分析为HCV组装和成熟的难以捉摸的机制提供了重要的见解。
The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 500 Å in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical and antigenic features in spite of the varying sizes of the particles. 3D reconstructions were obtained from HCV-LPs with the same size as the HCV virions in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a ‘fishbone’ configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3- and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.