Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography

Abstract

Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus) and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T=12 quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T=12 icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.

FIG. 1.

Morphology of RVFV MP-12 particles revealed by electron microscopy. (A) Negative-stain TEM micrograph showing pleomorphic RVFV MP-12 particles. (B) Negative-stain TEM micrograph showing spherical RVFV MP-12 particles with a distinct surface structure composed of morphological units with a central cavity. (C) Histogram representing the size distribution of negatively stained RVFV MP-12 particles, with an average diameter of 95 ± 9 nm (n = 84). The scale bar represents 100 nm.

FIG. 2.

Cryo-ET analysis and preliminary assessment of tomograms. (A) Slice from a tomogram of vitrified-hydrated RVFV MP-12 particles corresponding to the tilt stage position normal to the electron beam. Vesicles present in this tomogram are labeled “V.” An example of a distorted particle can be seen in close contact to the vesicle in the upper right corner (labeled with an asterisk). (B to D) Top (B), center (C), and bottom (D) slices from a tomogram of an isolated virus particle. This particle is identified by the black box in panel A. In the top slice, individual capsomeres are visible and indicated by the arrow. In a central slice, distinct densities corresponding to the three RNP complexes were not identified. In a bottom slice, bridging densities among capsomeres are indicated by the arrow. (E) Histogram representing the size distribution of frozen-hydrated RVFV MP-12 particles, with an average diameter of 103 ± 2 nm (n = 46). The scale bar represents 100 nm.

FIG. 3.

Structure of RVFV MP-12. Images were color coded according to radial distance. All particles are oriented along the threefold axis. (A) Shaded isosurface representation of RVFV MP-12 reconstructed from tilt series data at a 7.5-nm resolution. The structure was generated by averaging data for 46 individual particles extracted from three tomograms. The missing-wedge effect was greatly reduced because individual particle tomograms adopted different random orientations. Pentons and hexons are indicated by the arrows. (B) Cut-away view of the averaged RVFV MP-12 structure. No distinct densities were observed in the core of the particle that could be interpreted as RNP complexes. (C) Stereographic projection of the threefold (κ = 120°) self-rotation function calculated for the map reconstructed from tomographic data. The number and relative positions of peaks corresponded to those expected for an icosahedral particle. The peaks were sharp, implying strong symmetry relationships in the structure. The scale bar represents 50 nm.

FIG. 4.

RVFV MP-12 glycoprotein spike organization on an icosahedral lattice. Images were color coded according to radial distance. All particles are oriented along the threefold axis. (A) Shaded isosurface representation of RVFV MP-12 after the imposition of icosahedral symmetry onto the averaged tomogram shown in Fig. 3A. The icosahedral asymmetric unit is indicated by the black triangle. (B) Interior of the virus particle after the imposition of icosahedral symmetry. The central disordered RNP region was removed, thus exposing the inner surface of the lipid bilayer. (C) Central section through the map after the imposition of icosahedral symmetry as described in the legend to panel A. The dotted circles enclose the gap (white density) between the glycoprotein shell and the RNP core (gray density). Protein densities are represented in black. (D) Radial projection of the map after the imposition of icosahedral symmetry. Bridging densities among capsomeres were visible. Protein density is represented in black. The scale bar corresponds to 50 nm.