Structural analysis of pleomorphic capsid-like scaffold of Poxvirus using cryo-electron microscopy

使用冷冻电子显微镜对痘病毒多形性衣壳样支架进行结构分析

• 批准号: 21K06039
• 负责人: HYUN Jaekyung
• 金额: $ 1.5万
• 依托单位: Okinawa Institute of Science and Technology Graduate University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21K06039/
• 关键词: Cryo-electron micrscopy; Cryo-electron tomography; Icosahedral symmetry; Protein self-assembly; Virus capsid; Virus scaffold; Poxvirus; Cryo-electron microscopy

Recombinant Vaccinia virus scaffolding protein (D13) was successfully expressed and purified using bacterial expression system.Using cryo-electron microscopy (cryo-EM) single particle analysis, the structure of D13 trimer and doublet of the trimers were determined at 1.9 angstrom and 3.9 angstrom resolution, respectively. The structure and subsequent mutagenesis analysis allowed us to find the critical amino acid residues that are essential for D13 self-assembly into virion scaffold.D13 was subjected to self-assembly in vitro by altering salt concentration in the buffer, and we found that low salt concentrations below 150mM NaCl induce D13 self-assembly. The structure of resulting tubular assembly product was determined at 3.9 angstroms resolution. From the structure, we found that sextet of D13 trimers were arranged in a fashion similar to capsomers of icosahedral virus capsid. Also, cryo-electron tomography (cryo-ET) of immature virion-like particles showed that distinct inter-trimeric torsion induces continuous curvature of the scaffold, and renders a unique semi-regular morphology.The study revealed a novel assembly mechanism of immature Vaccinia virus that exemplifies a deviation from ideal quasi-symmetry proposed by Casper and Klug in 1960s. Also, identification of structural features of D13 in its self-assembly will contribute to the development of anti-viral treatments agains potential outbreak of poxvirus diseases.The study is being prepared for submission to a high-profile Journal.

利用细菌表达系统成功表达和纯化了重组痘苗病毒支架蛋白（D13）。采用低温电子显微镜（cryo-EM）单粒子分析，分别在1.9埃和3.9埃的分辨率下确定了D13三聚体的结构和三聚体的双重结构。结构和随后的诱变分析使我们找到了D13自组装成病毒粒子支架所必需的关键氨基酸残基。通过改变缓冲液中的盐浓度，D13在体外进行了自组装，我们发现低于150mM NaCl的低盐浓度诱导了D13的自组装。在3.9埃的分辨率下确定了管状组件产品的结构。从结构上，我们发现D13三聚体的六聚体的排列方式类似于二十面体病毒衣壳的衣壳体。此外，未成熟病毒粒子样粒子的低温电子断层扫描（cryo-ET）显示，明显的三聚体间扭转诱导支架的连续曲率，并呈现独特的半规则形态。该研究揭示了一种新的未成熟牛痘病毒组装机制，这种机制偏离了Casper和Klug在20世纪60年代提出的理想准对称。此外，确定D13自组装的结构特征将有助于开发针对痘病毒疾病潜在爆发的抗病毒治疗方法。这项研究正准备提交给一家知名杂志。

项目成果

Structural analysis of pleomorphic poxvirus assembly scaffold

多形性痘病毒组装支架的结构分析

• 发表时间: 2021
• 作者: Tsugita Atsushi;Uehara Shiro;Matsui Takashi;Yokoyama Takeshi;Ostash Iryna;Deneka Maksym;Yalamanchili Subbarao;Bennett Clay S.;Tanaka Yoshikazu;Ostash Bohdan;Jaekyung Hyun
• 通讯作者: Jaekyung Hyun

A capsid with a twist: Mechanism of pleomorphic immature poxvirus scaffold assembly

扭曲的衣壳：多形性未成熟痘病毒支架组装机制

• 发表时间: 2021
• 作者: Ito Hiroaki;Matsui Takashi;Konno Ryo;Itakura Makoto;Kodera Yoshio;Jaekyung Hyun
• 通讯作者: Jaekyung Hyun