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

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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.