Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Abstract

Bunyaviridae is a large family of viruses that have gained attention as "emerging viruses" because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or "spikes," were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.

Fig. 1.

UUKV particle morphology. (a and b) Low-magnification electron cryomicroscopy images of UUKV samples at pH 7 (a) and pH 6 (b). Both images show a representative area of the carbon support film with four 2-μm holes and vitrified virus sample. One virion is indicated with an arrowhead in a. At pH 7, the virions were distributed evenly, but at pH 6 they aggregated heavily. (Scale bars, 1 μm.) (c) A 5-nm-thick slice through a 3D tomogram of the pH7 sample is shown. The interior of UUK virions is visible, revealing the membrane and RNP density (black asterisk). Similar-appearing density, possibly leaked from the particles, was also identified (white asterisk). A few representative spikes around the UUKV particles are indicated (arrowheads). Some particles show deformations from a spherical shape, and often the membrane has a negative curvature (arrows). (Scale bar, 100 nm.)

Fig. 2.

Size histogram for UUKV particles. The maximum diameter of particles from the pH7 sample (n = 165, 92%) followed a normal distribution (mean = 125 nm, σ = 6 nm).

Fig. 3.

pH-dependent conformational differences in the glycoprotein spikes. (a) Individual particles with icosahedral symmetry applied are shown along the icosahedral 2-fold axis of symmetry. Representative particles from three different types of samples are shown, as indicated above the columns: pH7 was buffered at pH 7 and fixed with glutaraldehyde; pH7* was fixed with glutaraldehyde without buffering; pH6 was buffered at pH 6 before fixation. Each column represents data from a single particle. Isosurfaces were rendered at 1.5σ above the mean density. (Scale bar, 100 nm.) (b) Consecutive 8-nm-thick slices, 24 nm apart, are shown for the particles displayed in a. Within each panel, the icosahedrally symmetrized version of the particle is shown on the left, with the unsymmetrized, denoised particle on the right for comparison. The top row shows the central section. Arrows indicate RNP interactions with the membrane. (Scale bar, 100 nm.) (c) Close-ups from b (central section, top row) show the conformation of the spike in more detail. A cavity at the base of the spike is indicated with an arrow in pH7 and pH6 particles. (Scale bar, 50 nm.)

Fig. 4.

Glycoprotein spike organization. Shown are virions with spikes in tall conformation (a) and in flat conformation (b). Insets show close-ups of the area indicated with dashed lines. Flat spikes have a barrel-like appearance, whereas taller spikes have a more pointed appearance. The relationship between the five-coordinated positions (indicated with a pentagon) and six-coordinated positions (numbered 1–3) is consistent with T = 12 triangulation (43). Bridging densities (indicated by arrows) are present between the spikes at every position and in both conformations. Isosurfaces were rendered at 1.5σ above the mean density. (Scale bar, 100 nm.)

Fig. 5.

Radial density distribution. The radii for the RNPs, membrane, and spike protein layer are indicated for pH6 and pH7 samples. The RNP is preferentially located at three radii, as indicated by the three density maxima.

Fig. 6.

Composite model of the UUK virion in a native state. During entry to the host cell via the endocytotic pathway, the glycoprotein spikes (blue, tall conformation) are thought to change their conformation to facilitate fusion between the viral membrane (yellow) and a host endosomal membrane and, further, to release the RNPs (brown) into host cell cytoplasm. A part of the glycoprotein shell, and a slightly smaller part of the membrane, are removed to reveal the virion interior. The glycoprotein spikes and the membrane are rendered at 1.5σ and the RNP at 1.0σ above the mean density. The resolution of the reconstruction is 5.9 nm. For visualization, the RNP density was denoised.