Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins

Abstract

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.

FIG. 1.

Frozen-hydrated samples of Aura_T (A) and Aura_B (B) viruses. Bar, 1,000 Å.

FIG. 2.

Image reconstruction of Aura_T virus at 17-Å resolution, viewed along a twofold axis. (A) Surface-shaded representation. The yellow triangle demarks one icosahedral asymmetric unit. (B) Surface-shaded representation as in panel A but with the front half removed to reveal interior features. Arrows identify putative transmembrane connections. Aura virus exhibits a multilayered structure similar to that of SINV, which consists of glycoproteins (blue), a lipid bilayer (green), nucleocapsid proteins (orange), and the genome (pink). (C) Electron density map of an equatorial section. Icosahedral twofold (i2), threefold (i3), quasi-threefold (q3), and fivefold (i5) axes are labeled in panels A and C. Bar, 200 Å.

FIG. 3.

Comparison of Aura_T, Aura_B, SINV, and RRV reconstructions. Surface-shaded representations of the exteriors (top row), spikes (second row) (threefold [i3] and quasi-threefold [q3] axes), and equatorial cross sections (third row) from Aura_T and Aura_B, SINV, and RRV allow similarities and differences between these reconstruction maps to be compared. Reconstruction maps of Aura_T, Aura_B, and SINV are remarkably similar, whereas the RRV spike differs both on its exterior and its interior, where a cavity is present (see arrows on RRV cross section). The two panels on the bottom left are the central sections of the Aura_T−Aura_B and Aura_T−SINV difference maps. The bottom right panel plots the correlation coefficients between different pairs of reconstructions (Aura_T−Aura_B, Aura_T−SINV, and Aura_T−RRV) as a function of radius. The color bar at the top of the panel coincides with the color scheme used in the equatorial cross sections (third row): viral genome (pink), nucleocapsid core (orange), lipid membrane (green), and envelope glycoproteins (blue).

FIG. 4.

Stereo view of the electron density maps of Aura virus (red) and SINV (blue), displayed at a high contour level, with the capsid appearing at the bottom and the glycoprotein at the top. The finger-like projections pointing downwards from the glycoproteins and upward from the capsid, and corresponding to the transmembrane densities of Aura and SINV, are indistinguishable at the upper surface of the membrane but have slightly different orientations at the lower surface.

FIG. 5.

Stereo views of the Aura virus glycosylation sites. (A) Asymmetric unit of the Aura virus glycoproteins. Four SFV E1 molecules (blue) were fitted into the Aura_T density map at the lower portion of the spike and in the skirt (violet). The Aura virus carbohydrate densities are shown and were determined by generating difference maps between Aura_T and each of the four SINV deglycosylation mutants (17). The Aura_T carbohydrate densities are colored (E1-139 in brown, E1-245 in red, E2-197 in light green, and E2-319 in magenta). (B) Electron density of Aura virus and the shift in position between the Aura carbohydrate densities and those of SINV (green for E1-139 and blue for E2-318). The broken lines indicate possible pairs of Aura E1-139 and E2-319 carbohydrate sites which belong to one heterodimer (assignment I in Fig. 6).

FIG. 6.

Distances between E1-139 and E2-319 sites in the asymmetric unit in Aura virus. Distances are given in angstroms. The standard deviation (SD) for each assignment is also given. d_B1∗-A1, distance between B1∗ and A1.