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

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)，并利用低温电子显微镜(Cryo-EM)单颗粒分析，分别在1.9埃和3.9埃分辨率下测定了D13三聚体和二聚体的结构。结构和随后的突变分析使我们能够找到D13自组装到病毒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进行D13的体外自组装，我们发现低于150 mM的盐浓度可以诱导D13的自组装。最终得到的管状组装产物的结构以3.9埃的分辨率确定。从结构上我们发现，D13三聚体的六个三聚体以类似于二十面体病毒衣壳的方式排列。此外，未成熟病毒粒子的冷冻电子断层扫描(Cryo-ET)显示，明显的三聚体间扭曲导致支架连续弯曲，并呈现独特的半规则形态。研究揭示了未成熟痘苗病毒的一种新的组装机制，该机制偏离了Casper和Klug在20世纪60年代提出的理想准对称性。此外，识别D13自组装的结构特征将有助于开发针对可能爆发的痘病毒疾病的抗病毒治疗。这项研究正在准备提交给一份备受瞩目的杂志。

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.