Electron cryo-microscopy and single-particle averaging of Rift Valley fever virus: evidence for GN-GC glycoprotein heterodimers

Abstract

Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins G(N) and G(C), organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either G(N) and G(C) homodimers or G(N)-G(C) heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a G(N)-G(C) heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens.

FIG. 1.

Electron microscopy of RVFV. (A) Electron microscopy image of purified virus particles stained with 2% uranyl acetate. (B) Examples of views along twofold (left), threefold (middle), and fivefold (right) axes of icosahedral symmetry for stained particles. (C) Electron cryo-microscopy image of plunge-frozen, unstained virus particles. Scale bars, 100 nm.

FIG. 2.

Three-dimensional icosahedral reconstruction of RVFV. (A) Radially colored isosurface representation of the reconstruction. Glycoprotein cylinders are in blue, the glycoprotein base layer is in green, and the membrane and RNP density are in brown. One five-coordinated position (pentagon) and three different six-coordinated positions (hexagons 1, 2, 3) are indicated. In the lower left part of the panel, we removed the front half of the reconstruction to reveal the capsomers from the side (a single capsomer is indicated with a red oval). The isosurface was rendered at 1.3σ above mean density. Scale bar, 50 nm. (B) Central cross section through the density (black, high density; white, low density). The indicated radii correspond to the glycoprotein shell, the membrane, and the RNPs. A shell of RNP density, closely following the shape of the membrane, is indicated with an asterisk. The membrane is distorted from a round shape, making kinks toward the bases of glycoprotein cylinders (some indicated with arrows). The same capsomer as in panel A is indicated with a red oval. Scale bar, 50 nm. (C to E) Close-ups of cross sections showing transmembrane densities (indicated with arrows). Scale bar, 10 nm (C to E). The cross sections in panels B to E are 0.4 nm thick.

FIG. 3.

Resolution and radial-density distribution of the RVFV reconstruction. (A) A Fourier shell correlation as a function of spatial frequency was calculated between two test reconstructions, calculated from two independent half-sets of the data. It shows correlation above the 0.5 threshold up to 2.2-nm resolution. (B) Radially averaged density (solid line) and resolution (dotted line) are plotted as a function of the radius. The radii corresponding to the glycoprotein shell, the membrane, and the RNPs are indicated. The shell of RNP density proximal to the membrane is indicated with an asterisk.

FIG. 4.

Comparison of the external glycoprotein densities in different positions of the lattice. Shown are top views (top row) and side views (bottom row) of the densities extracted from the three quasi-sixfold positions (hexagons 1, 2, and 3) and from the fivefold position (pentagon). A cylindrical mask extending to halfway between neighboring densities was used in the extraction. The fivefold cylinder is composed of subunits shaped similarly to subunits of the quasi-sixfold cylinders, suggesting that there is only one type of structural building block in the glycoprotein shell. The isosurface was rendered at 1.3σ above mean density and radially colored, as in Fig. 2A. Scale bar, 10 nm.

FIG. 5.

Molecular organization of the glycoprotein lattice. (A) A closed surface with a shape between a perfect sphere and an icosahedron was fitted to traverse glycoprotein cylinders, and the density values were mapped on the surface (black, low density; white, high density). Approximate radii for the surfaces are indicated. The insets show close-up views of one five- and one neighboring six-coordinated position at each radius. Surfaces at radii of 48 to 50 nm show cylinders of glycoproteins at five-coordinated and six-coordinated positions of the T=12 lattice. At radii of 45 to 47 nm, quasi-twofold contacts between the capsomers become apparent. At radii of 41 to 44 nm, densities from three neighboring capsomers come together at quasi-threefold positions to form triangular densities, connecting the capsomers to the underlying membrane. Surfaces at radii of 39 and 40 nm traverse the outer leaflet of the lipid bilayer and the space between the two leaflets, revealing the punctate pattern of transmembrane densities. The relative positions of the transmembrane densities are indicated with red for surfaces at radii of 34 to 41 nm to correlate their positions with the other features of the reconstruction. One membrane patch of the outer leaflet with a low curvature and surrounded by the transmembrane densities is indicated with an asterisk at 40 nm. Scale bar, 50 nm. (B) Part of the RVFV T=12 lattice is illustrated as a schematic in the same orientation as in panel A. A five-coordinated cylinder (pentagon) and six-coordinated cylinders (1, 2, and 3) are indicated. The positions of some of the quasi-twofold contacts (rectangles) and quasithreefold contacts (triangles) are also indicated.