Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers Juha T. Huiskonen, Anna K. Överby, Friedemann Weber and Kay Grünewald J. Virol. 2009, 83(8):3762. DOI: 10.1128/JVI.02483-08. Published Ahead of Print 4 February 2009.
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JOURNAL OF VIROLOGY, Apr. 2009, p. 3762–3769 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.02483-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers䌤 ¨ verby,3 Friedemann Weber,3 and Kay Gru Juha T. Huiskonen,1,2* Anna K. O ¨newald1 Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany1; Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland2; and Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79008 Freiburg, Germany3 Received 3 December 2008/Accepted 23 January 2009
reticulum. Formation of a heterodimeric complex of the glycoproteins is required for targeting to the Golgi apparatus, since only GN has the Golgi localization signal (7, 14). GN and GC are type I transmembrane proteins. They both traverse the lipid bilayer, and the C-terminal tails are internal to the virion. Unlike other negative-stranded RNA viruses, bunyaviruses lack a matrix protein (37). Thus, the glycoprotein C-terminal tails are thought to interact directly with the RNPs during budding to ensure the proper packaging of the RNPs (29, 30). In RVFV, the M segment encodes two proteins in addition to the two glycoproteins GN (54 kDa) and GC (56 kDa): the nonstructural protein NSm and an additional 78-kDa protein, which is a minor component of the virion (13). The glycoproteins GN and GC have been shown to form heterodimers in cells infected with RVFV (13) and with other phleboviruses, such as Uukuniemi virus (UUKV) (32) and Punta Toro virus (6). However, as reassociations of the glycoproteins during virus maturation cannot be ruled out, the multimeric status of the glycoproteins in the virions is not well established. In fact, there exist conflicting reports about isolation of both heterodimers (32) and homodimers (36) from Triton X-100-solubilized UUKV virions. The structure of RVFV virions studied recently using electron cryo-tomography revealed five- and six-coordinated glycoprotein capsomers organized on a T⫽12 lattice (10). However, the limited resolution (6.1 nm) impeded determination of the compositions of the capsomers. It remained unclear whether a single structural unit composed of both GN and GC forms both the five- and six-coordinated glycoprotein capsomers or whether GN alone forms some capsomers and GC alone forms the other capsomers (10). In addition, the organization of RNPs inside the virions could not be addressed. Knowledge of the structural
Rift Valley fever virus (RVFV) is a mosquito-transmitted virus causing diseases in humans ranging from mild flu-like symptoms to severe hemorrhagic fever (12). Since its first isolation in 1930 (9), several large outbreaks have been identified in Sub-Saharan Africa, where it is endemic, and also in neighboring regions, such as Egypt and the Arabian Peninsula (2, 5). Humans can acquire RVFV through mosquito bites or exposure to infected animal material. In cattle, RVFV causes abortions and high levels of neonatal mortality. Thus, RVFV is a medically and economically significant pathogen. RVFV is a member of the genus Phlebovirus within the family Bunyaviridae. This family is divided into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus (25). Common to all members of the family Bunyaviridae is the characteristic that the virions possess a singlestranded, tripartite RNA genome core enclosed by a lipid bilayer and a shell of glycoproteins (37). Bunyaviruses replicate in the cytoplasm, and nascent virions assemble by budding into the lumen of the Golgi apparatus (35). Bunyaviruses encode four structural proteins from the three genome segments: the L segment encodes the RNA-dependent RNA polymerase (L), the M segment encodes the two membrane glycoproteins (GN and GC), and the S segment encodes the nucleocapsid protein (N) (37). The N protein coats the genome segments to form ribonucleoproteins (RNPs). The two glycoproteins are translated as a polyprotein, which is cleaved in the endoplasmic * Corresponding author. Mailing address: Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany. Phone: 49 89 8578-2632. Fax: 49 89 8578-2641. E-mail:
[email protected]. 䌤 Published ahead of print on 4 February 2009. 3762
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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 GN and GC, organized on a Tⴝ12 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 GN and GC homodimers or GN-GC 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 GN-GC 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.
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MATERIALS AND METHODS Cell culture and virus purification. Vero cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and maintained in medium with 2% serum. The Vero cells were infected with RVFV strain Clone 13 at a multiplicity of infection of 0.01, and the supernatant containing virions was collected 72 h postinfection. The supernatant was clarified by low-speed centrifugation (6,000 rpm) for 10 min before the pH 6.0 or 7.4 was adjusted with phosphate buffer (final concentration, 50 M). The virus was fixed with glutaraldehyde (final concentration, 0.5%) and concentrated through a sucrose cushion (20% [wt/vol]) in TN buffer (50 mM Tris-HCl, pH 6.0 or 7.4, 100 mM NaCl) at 100,000 ⫻ g for 1 h. The virus pellet was resuspended in TN buffer. Electron cryo-microscopy and image processing. A 3-l aliquot of freshly prepared virus suspension at pH 6.0 or pH 7.4 was pipetted on a glow-discharged holey carbon-coated electron microscopy grid (C-flat; Protochips), excess suspension was blotted with a filter paper, and the sample was vitrified by plunging it rapidly into liquid ethane. Electron cryo-microscopy was performed using a Tecnai F20 microscope (FEI) operated at 200 keV and equipped with a 4,000-by-4,000 charge-coupled-device camera (Eagle; FEI). Low-dose images (20 e⫺/ A2) were taken with SerialEM (23) and 1- to 3-m underfocus at a nominal magnification of ⫻80,000, giving a calibrated pixel size of 0.13 nm. Images were computationally down-sampled by a factor of 3 to give a final pixel size of 0.40 nm. Images free from drift and astigmatism were used to extract views for threedimensional reconstruction of the virion at pH 6.0 and pH 7.4. Virus particles were automatically localized in the electron micrographs with ETHAN (20), and subimages of single viral particles were boxed in EMAN (22). One hundred thirty particles extracted from 42 micrographs were used for the pH 6.0 reconstruction and 267 particles extracted from 111 micrographs for the pH 7.4 reconstruction. Bsoft was used in further image-processing tasks unless otherwise stated (17). Density values in the images were normalized to a common average value and standard deviation (). Contrast transfer function parameters were determined, and images were corrected for the contrast reversals by flipping the phases at appropriate spatial frequencies. Orientations and origins for particles were determined using a modified version of the original polar Fourier transform algorithm (1). Three-dimensional reconstructions were computed with a FourierBessel algorithm (8, 11). The previously published model of UUKV (28) was used as a starting model in model-based refinement of particle origins and orientations, and reconstruction processes were iterated until no improvement was observed in the resolution. Icosahedral symmetry had been detected in RVFV particles previously (10) and was imposed here on the reconstructions. The resolution of each reconstruction was evaluated using two test reconstructions, both calculated from independent half-sets of particle views to 0.8-nm resolution. Fourier shell correlation was calculated between the glycoprotein shells of the two test reconstructions, and the resolution was determined using a threshold of 0.5. In addition, the radial resolution was determined for 4-nm-thick shells at every 0.4 nm using Fourier shell correlation and the same threshold. The final pH 6.0 and pH 7.4 reconstructions were calculated to 2.3-nm and 2.0-nm resolutions, respectively. A difference map was calculated between the pH 6.0 reconstruction and pH 7.4 reconstruction at 3.0-nm resolution.
For comparison, glycoprotein densities external to the membrane were masked out from the pH 7.4 reconstruction. Glycoprotein cylinders were computationally rotated to align their cylindrical axes on the icosahedral twofold axis of symmetry. The rotations required for three glycoprotein cylinders situated on the icosahedral-symmetry axis were known from the icosahedral symmetry (see Fig. 2 and 5, positions 2, 3, and 4), but the rotation required for the cylinder situated off the symmetry axis (see Fig. 2 and 5, position 1) had to be determined. Density extracted from the twofold position acted as a template (see Fig. 2 and 5, position 2) in an orientation search based on cross-correlation of the two volumes. Three-dimensional visualizations were done in UCSF Chimera (34). To estimate the molecular masses of the glycoprotein capsomers, the RNP and membrane were removed from the pH 7.4 reconstruction with a spherical mask (radius, 42 nm). The volume was calculated in EMAN (22). We tested different threshold values for the molecular surface ( above the mean density). The average density for a solvated protein (1.35 g/ml) was assumed to calculate the mass (24). Density map accession numbers. The reconstructions have been submitted to the EM Data Bank with accession codes EMD-1549 (pH 6.0) and EMD-1550 (pH 7.4).
RESULTS Electron microscopy of purified RVFV virions. Icosahedral symmetry was detected in the outer glycoprotein shells of UUKV particles in a recent study using electron cryo-tomography (28). This suggested that other phlebovirus virions might also be icosahedrally symmetric, rather than being pleomorphic, as previously thought. Furthermore, it raised the possibility of exploiting a single-particle averaging approach, where multiple virions are computationally averaged together and icosahedral symmetry is imposed (1). When applicable, this approach is beneficial, since it typically yields a higher resolution than is possible by electron cryo-tomography. To study whether this averaging approach was suitable for RVFV, we first analyzed the morphology of purified, negatively stained RVFV virions using electron microscopy. The particles had nearly uniform sizes and a roughly round shape in projection images (Fig. 1A). More strikingly, some of the RVFV particles displayed clear twofold, threefold, and fivefold symmetric views, arguing for the presence of icosahedral symmetry (Fig. 1B). These results were in agreement with a recently published tomographic reconstruction in which icosahedral symmetry was detected (10). We used electron cryo-microscopy to visualize purified, unstained RVFV virions (Fig. 1C). Electron cryo-microscopy projection images revealed round particles of homogeneous sizes and shapes (Fig. 1C). This was in contrast to an earlier electron cryo-microscopy study of La Crosse bunyavirus, where round particles with heterogeneous sizes were observed (38). Fixation with 0.5% glutaraldehyde prior to purification was required to reduce excessive aggregation of virus particles occurring in the unfixed sample (not shown). Fixation not only preserves fragile complexes during purification, but also changes the surface properties of the particles and thus affects particle aggregation (19). Fixation likely also helped to preserve RVFV particle morphology in negative staining (Fig. 1A), since unfixed RVFV particles are commonly seen to be distorted (10). Three-dimensional reconstruction of RVFV virions revealed a multilayered structure. We calculated a three-dimensional reconstruction of the virion by averaging together particle projection images and applying icosahedral symmetry (Fig. 2). Micrographs were recorded in those areas of the sample that
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composition of RVFV glycoprotein capsomers and of the RNP organization is crucial in understanding the assembly and infection mechanisms of this important pathogen. Here, we have studied the structure of the RVFV virion using electron cryo-microscopy combined with three-dimensional image reconstruction and single-particle averaging. The structure was solved to 2.2-nm resolution. The structure revealed 110 cylinder-shaped glycoprotein hexamers and 12 pentamers organized on an icosahedral T⫽12 lattice. Only one type of structural unit was detected in the capsomers. This structural unit most likely corresponds to a GN-GC heterodimer. Six structural units form the hexamers, and five form the pentamers. Transmembrane densities, each corresponding most likely to several glycoprotein transmembrane helices, were seen to modulate the curvature of the bilayer. Furthermore, the organization of the membrane-proximal RNP density correlated with the positions of these transmembrane densities.
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showed only mild particle aggregation (Fig. 1C). We included 267 out of 472 particle images (57%) from 111 micrographs in the reconstruction. The resolution of the reconstruction was 2.2 nm, as determined for the glycoprotein shell by Fourier shell correlation, with a 0.5 cutoff (Fig. 3A). Sample aggregation may distort some of the particles from perfect icosahedral symmetry, thereby limiting the achievable resolution. The shape of the virion is nearly spherical, with the diameter varying only slightly from 101 nm to 106 nm, as measured from icosahedron edge to edge and vertex to vertex, respectively. The virion surface is entirely covered by a shell of 122 glycoprotein capsomers. The capsomers resemble hollow cylinders when seen from the outside of the particle (Fig. 2A). The cylinders are situated at the five-coordinated (Fig. 2A, pentagon) and at three types of six-coordinated (Fig. 2A, positions 1, 2, and 3) positions of a T⫽12 icosahedral lattice, as described previously (10). Twelve cylinders are situated at the fivefoldsymmetry axis of the icosahedral lattice (Fig. 2A, pentagon), 60 cylinders are off the symmetry axis (Fig. 2A, hexagon 1), 30 cylinders are at the twofold-symmetry axis (Fig. 2A, hexagon 2), and 20 cylinders are at the threefold-symmetry axis (Fig. 2A, hexagon 3). The cylinders at five-coordinated positions are slightly smaller (outer diameter, 14 nm) than the cylinders at the six-coordinated positions (outer diameter, 16 nm). Under the glycoprotein shell, two leaflets of the lipid bilayer are clearly discernible (Fig. 2B). Strikingly, the membrane is not completely round but instead makes kinks toward the glycoproteins (Fig. 2B). This is likely due to the presence of transmembrane segments of glycoproteins at these positions, sometimes visible as transmembrane densities (Fig. 2C to E). Furthermore, this is reminiscent of many other membranecontaining viruses, such as dengue virus, in which transmembrane domains modulate the curvature of the bilayer (18, 21, 39). A layer of RNP is located proximal to the inner leaflet of the membrane and closely follows the shape of the membrane (Fig. 2B). We calculated a radially averaged density distribution of the RVFV virion that clearly showed the glycoprotein shell, lipid bilayer, and RNP density (Fig. 3B). The glycoproteins protrude 9 nm from the membrane surface and are situated at 42- to 51-nm radii. The glycoprotein shell is divided into two peaks at 42 to 46 nm and at 47 to 51 nm, reflecting the domain organization of the glycoprotein capsomers (see below). The lipid bilayer is visible at the 35- to 42-nm radii, with the strongly scattering phospholipid head groups likely causing the two density maxima at the 36- and 40-nm radii. A plot of the resolution against the particle radius revealed a radially anisotropic resolution in the reconstruction (Fig. 3B). This was due to variable degrees of order in the different components of the virion. While the outer part of the reconstruction—the glycoprotein shell and membrane—has the highest resolution of 2.1 to 2.3 nm, the inner RNP density is unresolved. In conclusion, the RNP density is not icosahedrally ordered. However, the RNP density is not completely evenly distributed either. Rather, there is a distinct layer proximal to the membrane at 32- to 34-nm radius (Fig. 3B). This distribution likely reflects interactions between the C-terminal tails of the glycoproteins and RNPs, as suggested for UUKV (28, 29). The distance between this membrane-proximal RNP layer and the membrane is 4.4 nm, as measured from peak to peak.
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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.
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Structures of the glycoprotein capsomers. To compare the structures of the four different classes of glycoprotein capsomers (Fig. 2A, pentagon and hexagons 1, 2, and 3), their respective densities were extracted from the reconstruction (Fig. 4). Reliable demarcation of molecular boundaries between the
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 fivecoordinated 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 1.3 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.
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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.
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capsomers was not possible due to the limited resolution, and thus, we used a cylindrical mask as an approximation. Densities extracted from all three six-coordinated positions were strikingly similar to each other, showing the same structural features arranged in quasi-sixfold symmetry (Fig. 4, hexagons 1, 2, and 3). Furthermore, similarly shaped subunits form the capsomers at five-coordinated (Fig. 4, pentagon) and at six-coordinated (Fig. 4, hexagons 1, 2, and 3) positions. Organization of the glycoprotein shell. The complex organization of the RVFV glycoprotein shell is shown in Fig. 5. This shell can be divided conceptually into four different layers: (i) glycoprotein cylinders (Fig. 5A, radii of 48 to 50 nm), (ii) intracylinder contacts (Fig. 5A, radii of 45 to 47 nm), (iii) triangular membrane connections (Fig. 5A, radii of 41 to 44 nm), and (iv) transmembrane densities (Fig. 5A, radii of 39 to 40 nm). In the first, outermost layer, the glycoprotein cylinders at six-coordinated positions (Fig. 5B, hexagons) show a quasisixfold symmetry, with a pattern of six two-lobed structural units (Fig. 5A, radii of 48 to 50 nm). The cylinders at fivecoordinated positions (Fig. 5B, pentagon) are composed of five similar two-lobed structural units. In some, but not in all, quasi-twofold positions, neighboring cylinders are connected to each other via thin, bridging densities (Fig. 5A, 50 nm). In the second layer, the glycoprotein cylinders form extensive intracylinder contacts at each of the quasi-twofold positions (Fig. 5A, radii of 45 to 47 nm, and B, rectangles). Two spokes, one from each cylinder, bind together in a parallel fashion. In the third layer, the densities from the glycoprotein cylinders and spokes come together from three neighboring capsomers and form triangular densities (Fig. 5A, radii of 41 to 44 nm, and B, triangles). These densities connect the capsomers to the underlying membrane. In the fourth layer, the triangular membraneproximal densities split into a punctate pattern of transmembrane densities (Fig. 5A, radii of 39 to 40 nm) (see below).
The positions of transmembrane densities correlate with membrane curvature and RNP organization. Triangular densities were seen to connect the glycoproteins to the underlying membrane (Fig. 5A, radius of 41 nm). Both of the glycoproteins are expected to contain one most likely alpha-helical transmembrane segment (13). Thus, under each of the triangular membrane-proximal densities, transmembrane helices are expected to be present. At 2.2-nm resolution, it was not possible to identify single helices, which typically have a diameter of only 0.7 nm. However, visual inspection of the density map revealed several positions in which clear density was connecting the two leaflets of lipid bilayer (Fig. 2C to E and 5A, radius of 39 nm and inset). These transmembrane densities likely correspond to two or more transmembrane helices. As expected, the transmembrane densities are situated directly underneath the triangular membrane-proximal densities (Fig. 5A, radius of 41 nm). Furthermore, the organization of the transmembrane densities correlated with the observed kinks in membrane curvature. The transmembrane densities create a high membrane curvature by pulling the membrane toward the glycoprotein shell (Fig. 2C to E) and thus encircle membrane areas with a smaller curvature, as shown for the bilayer outer leaflet in Fig. 5 (radius, 40 nm). We correlated the positions of the transmembrane densities with the lateral distribution of membrane-proximal RNP density. The membrane-proximal RNP density is unevenly distributed. Strikingly, the RNP prefers areas directly under the transmembrane densities (Fig. 5A, radius of 34 nm). Although glycoprotein-RNP connections were not resolved here, the colocalization of RNP and glycoprotein transmembrane densities argues for a specific interaction between the glycoprotein cytoplasmic tails and RNP complexes. Glycoprotein heterodimers form the glycoprotein shell. Given the similarity in the subunits of five- and six-coordinated
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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 1.3 above mean density and radially colored, as in Fig. 2A. Scale bar, 10 nm.
Downloaded from http://jvi.asm.org/ on April 26, 2014 by PENN STATE UNIV 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. 3767
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DISCUSSION In this study, we have addressed the glycoprotein organization of the RVFV virion. The virion structure argues against possible heterodimer-to-homodimer transitions of viral glycoproteins during assembly or maturation. Rather, GN and GC are organized in the virion as heterodimers, just as they are expressed (13). These heterodimers form pentons and hexons and assemble on a T⫽12 icosahedral lattice. However, in the assembled glycoprotein shell, GN-GN and GC-GC contacts are also likely to occur. For example, at quasi-twofold positions, where the intracylinder contacts occur, homodimeric contacts are present (Fig. 5A and B, rectangles). This could explain the seemingly contradictory results for UUKV, another phlebovirus, where both homodimers (36) and heterodimers (32) have been extracted from the virions. Extraction of membrane proteins from virions could stabilize protein-protein interactions occurring only in the assembled state. Only two members of the family Bunyaviridae, RVFV and UUKV (both members of the genus Phlebovirus), have been structurally characterized using electron cryo-microscopy (10, 28). While the overall architectures of the two are the same (T⫽12), the glycoprotein capsomers differ in their morpholo-
gies, RNP interactions, and pH sensitivities. RVFV capsomers resemble hollow cylinders and protrude 9 nm from the membrane, whereas UUKV capsomers resemble spikes protruding 13 nm from the membrane (28). The glycoproteins of UUKV are considerably larger (GN ⫽ 75 kDa; GC ⫽ 65 kDa) than those of RVFV (GN ⫽ 54 kDa; GC ⫽ 56 kDa), which probably explains the larger glycoprotein capsomers of UUKV. The RVFV membrane-proximal RNP density was more ordered than the rest of the RNP. Although similar ordering was observed for UUKV, the RNPs seem to be less important for particle assembly in UUKV than in RVFV. UUKV glycoproteins are sufficient to form empty virus-like particles without the nucleocapsid protein (31). In the case of RVFV, glycoproteins alone are not sufficient for generating virus-like particles, but nucleocapsid protein, and potentially also RNA, are required (16). While the interaction between the RVFV N protein and RNA has not been investigated, in members of the genus Orthobunyavirus, the N protein is known to bind viral RNA, cRNA, and, to a lesser extent, also nonspecific cellular RNA (26, 27). Thus, it is possible that cellular RNA is also incorporated into RVFV virus-like particles. Considering these findings, we suggest that the interaction between the RNP and the glycoproteins stabilizes the RVFV virions or drives their assembly. This hypothesis is supported by the results of our study, which show that the positions of membrane-proximal RNP density and glycoprotein transmembrane densities are correlated. pH-dependent conformational changes have been suggested to play an essential role during bunyavirus entry in the endosome. These conformational changes have been reported to occur at pH 6.4 in the GC of La Crosse virus (15) and between pH 6.0 and 6.2 in the GC of UUKV (36). We have recently observed a conformational change of UUKV glycoprotein capsomers in virions at pH 6.0, studied using electron cryo-tomography (31). To study if pH-dependent conformational changes would also occur in RVFV, we analyzed samples prepared at pH 6.0, in addition to those prepared at pH 7.4 (see Materials and Methods). Unexpectedly, the difference map calculated for the two reconstructions did not reveal any significant differences (not shown). Further studies over a broader pH range are required to gain insight into possible pH-dependent structural changes and the fusion mechanism of RVFV. The view that bunyaviruses are pleomorphic, lacking welldefined shape, is now changing rapidly for the members of the genus Phlebovirus. Virions of this genus are likely to have icosahedral symmetry, as suggested earlier for UUKV and more recently for RVFV (10, 28). Here, we have demonstrated that the single-particle averaging approach exploiting icosahedral symmetry is feasible for studying the RVFV structure. The achieved 2.2-nm resolution allowed us to analyze the viral glycoprotein organization to a level that was not possible in an earlier tomographic reconstruction at a much lower resolution (6.1 nm) (10). Through improvements in sample purification and preservation, we expect the approach taken here to also be applicable to other phleboviruses at increasing resolutions. Electron cryo-microscopy and single-particle averaging, together with X-ray structure determination of phlebovirus glycoprotein structures, would enable more detailed understanding of the assembly and
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capsomers (Fig. 4 and 5), we reject the hypothesis that one of the glycoproteins alone would form a certain type of capsomer (e.g., the five-coordinated capsomers) and the other glycoprotein alone would form the rest of the capsomers (e.g., the six-coordinated capsomers). Rather, there is only one basic structural unit forming the glycoprotein shell. Six copies of this unit form each of the 110 six-coordinated capsomers, and five copies form each of the 12 five-coordinated capsomers. Thus, the total number of structural units is 720. The basic structural unit is most likely composed of both GN and GC, since they are both major components of the virion and are expected to be present in an equimolar ratio, as shown for UUKV particles (33). It is also likely that both GN and GC are exposed on the RVFV surface, since monoclonal antibodies against both GN and GC can neutralize RVFV infection (3). How many copies of GN and GC contribute, then, to the basic structural unit? The GN and GC external parts correspond to a molecular mass of ⬃45 kDa and ⬃51 kDa, respectively (4). To assess how many copies of GN and GC can fit in the reconstruction, we estimated the total mass of the glycoprotein shell from the reconstruction (see Materials and Methods). We carried out the estimation at different thresholds ( above the mean density) for the molecular surface. Commonly used thresholds for the molecular surface (1.3 to 2.0) corresponded to a molecular mass of 68 to 86 MDa. The mass calculated using a much lower threshold (0.8) was 100 MDa. This threshold is at the noise level of the reconstruction and thus provides an upper estimate for the molecular mass. The structural unit of a single GN-GC heterodimer would give a total calculated molecular mass of 69 MDa (720 heterodimers) for the glycoprotein external parts. A dimer of heterodimers would correspond to 138 MDa (1,440 heterodimers), a much greater mass than our upper estimate. Thus, our mass measurement is compatible only with the former assumption, and we conclude that 720 GN-GC heterodimers form the glycoprotein shell of RVFV.
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infection mechanism of these negative-stranded RNA viruses lacking a matrix protein. 20.
ACKNOWLEDGMENTS We acknowledge support from the Academy of Finland (114649 to J.T.H.), from the European Molecular Biology Organization (ALTF 820-2006 to J.T.H.), and from the Deutsche Forschungsgemeinschaft (GR1990/1-3 and -2-1 to K.G. and WE 2616/5-1 to F.W.). REFERENCES
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