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. 2012 Oct;86(20):11078-85.
doi: 10.1128/JVI.01390-12. Epub 2012 Aug 1.

Cryo-electron tomography of rubella virus

Anthony J Battisti  1 , Joshua D Yoder, Pavel Plevka, Dennis C Winkler, Vidya Mangala Prasad, Richard J Kuhn, Teryl K Frey, Alasdair C Steven, Michael G Rossmann
Affiliations

Cryo-electron tomography of rubella virus

Anthony J Battisti et al. J Virol. 2012 Oct.

Abstract

Rubella virus is the only member of the Rubivirus genus within the Togaviridae family and is the causative agent of the childhood disease known as rubella or German measles. Here, we report the use of cryo-electron tomography to examine the three-dimensional structure of rubella virions and compare their structure to that of Ross River virus, a togavirus belonging the genus Alphavirus. The ectodomains of the rubella virus glycoproteins, E1 and E2, are shown to be organized into extended rows of density, separated by 9 nm on the viral surface. We also show that the rubella virus nucleocapsid structure often forms a roughly spherical shell which lacks high density at its center. While many rubella virions are approximately spherical and have dimensions similar to that of the icosahedral Ross River virus, the present results indicate that rubella exhibits a large degree of pleomorphy. In addition, we used rotation function calculations and other analyses to show that approximately spherical rubella virions lack the icosahedral organization which characterizes Ross River and other alphaviruses. The present results indicate that the assembly mechanism of rubella virus, which has previously been shown to differ from that of the alphavirus assembly pathway, leads to an organization of the rubella virus structural proteins that is different from that of alphaviruses.

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Figures

Fig 1
Fig 1
Pleomorphic nature of rubella virus. (A) A tomographic reconstruction showing the cross-section of several rubella virions near their center. The virions are variable in size and shape. The black arrow indicates an approximately spherical virion and the white arrow indicates a tear-drop-shaped virion. A nearly cylindrical virion is also shown (inset right). The images were generated by averaging four planes of voxels and represent a thickness of 6.0 nm. High density is black, and the scale bar represents 50 nm. (B) The diameters of 51 spherical virions range from 57 to 86 nm and follow a normal distribution, with the largest fraction of virions having a diameter close to 70 nm.
Fig 2
Fig 2
Morphological characteristics. (A) Central sections through two spherical rubella (left panels) and two Ross River (right panels) virions. The black arrowhead indicates an outer shell of density for rubella virus, presumed to consist of the viral membrane with embedded E1 and E2 glycoproteins. The white asterisk indicates spike structures that extend up to 8 nm from the virion surface. The inner shell of density, or nucleocapsid, is indicated by the white arrowhead. The images were generated by averaging three planes of voxels and represent a thickness of 4.5 nm. High density is black, and the scale bar represents 25 nm. (B) Radial density distributions were calculated for the rubella and Ross River virus data. The solid curve represents an average of five spherical rubella virions with a diameter close to 70 nm. The dotted curve represents an average of five Ross River virions. The Ross River data shows a high and continuous density from the virion center out to a radius of about 15 nm (white bar), whereas a region of low density extends from the rubella virion center to the ∼14-nm inner radius of the nucleocapsid structure (black bar). Both rubella and Ross River virions have a gap, or region of low density, between the nucleocapsid and membrane. This gap is located at a radius of about 25 nm for rubella virus (black diamond), and about 18 nm for Ross River virus (white diamond). Densities were scaled such that the rubella and Ross River glycoprotein (outer shell) features would have equivalent density height. The standard errors in the density measurements were about 6 arbitrary units for the Ross River virus data and about 8 arbitrary units for the rubella virus data. The measurements were made with the same charge-coupled device detector at the same magnification. (C) Tomographic sections tangential to the surface of spherical rubella virions show parallel rows of E1-E2 glycoprotein spikes separated by 9.0 nm. The white arrows indicate pairs of equi-spaced rows of spikes. The spike structures are orthogonal to the plane of the page. Images were generated by averaging three planes of voxels and represent a thickness of 4.5 nm. High density is black, and the scale bar represents 25 nm. (D) Central sections through a pair of nonspherical rubella virions. The viral envelope and closely apposed nucleocapsid density are indicated by the black and white arrowheads, respectively. The images were generated by averaging three planes of voxels and represent a thickness of 4.5 nm. High density is black, and the scale bar represents 25 nm.
Fig 3
Fig 3
Phased self-rotation function search for 5-fold symmetry. (A) Stereographic projection of the κ = 72° phased self-rotation function search plotted as a function of ψ and φ for a Ross River virus subtomogram. (B) The results of an equivalent search for a rubella virus subtomogram. The virion subtomograms were placed in the most probable standard orientation using the methods described (see Results and Discussion, “Orientation search enforcing icosahedral symmetry”). The great circles meet at the locations of the icosahedral 2-folds and form pentagonal outlines centered at ψ and φ values that coincide with the 5-fold vertices. Values of ψ are indicated on the plot and the longitudinal demarcations represent 15° increments in φ. The contours are drawn in 0.5 standard deviation increments starting from 1.0. (C) The map (ρh, icos) generated by enforcement of icosahedral symmetry for the Ross River virus subtomogram evaluated in A. (D) Map generated by the enforcement of icosahedral symmetry for the rubella virus subtomogram evaluated in panel B. The black triangles indicate the icosahedral asymmetric unit, and the scale bars represent 25 nm. Maps are shown at 2.0 standard deviations above the mean density height.
Fig 4
Fig 4
Analysis and interpretation of E1-E2 glycoprotein spikes. (A) Tomographic section tangential to the membrane of an elongated virion showing several parallel rows of glycoprotein structure orthogonal to the long axis of the virion. The density was scanned along a line in the direction of the white arrow, parallel to the length of the virion. The scale bar represents 50 nm, and high density is black. (B) The density height measured across the repeating glycoprotein structure shows a regular 9.0-nm repeat. The spaces between rows alternate between higher and lower density height, indicating that rows of glycoprotein spikes are paired. The two halves of a pair are more strongly connected than the adjacent unpaired rows. (C) Model depicting a possible organization of E1 (red ellipsoids) and E2 (blue spheres). E2 may link paired rows of the more accessible E1 spikes. The top and bottom panels represent orthogonal views, and the curved gray bar represents the viral membrane.
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