In vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus

Abstract

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 A with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.

FIG. 1.

CryoEM density maps of RRV CLPs, WEEV CLPs, and the nucleocapsid core excised from the reconstruction of mature RRV. (A to C) Stereo diagrams, where only the top hemisphere of the map is shown. All maps are at 30 Å resolution and contoured at 2.5 σ. The white triangle represents one icosahedral asymmetric unit at a radius of 170 Å. The top vertex of the triangle is at the fivefold-symmetry axis. (A) The RRV CLP structure with density shown between radii of 150 and 210 Å. (B) The densities of both RRV CLP (blue; radii of 150 to 210 Å) and WEEV CLP (pink; radii of 180 to 210 Å). Note the quasi-symmetry present in both CLP reconstructions. (C) Superimposed densities of RRV CLP (blue; radii of 150 to 210 Å) and the nucleocapsid core from mature RRV (green; radii of 150 to 210 Å). (D) Central cross section along twofold axis of RRV CLP (blue) and WEEV CLP (pink), both from radii of 150 to 210 Å. (E) Same orientation as shown in panel D, with RRV CLP (blue) and the nucleocapsid core from the mature RRV particle (green).