Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus

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Science  05 Feb 2010:
Vol. 327, Issue 5966, pp. 689-693
DOI: 10.1126/science.1181766

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Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo–electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.

Vesicular stomatitis virus (VSV) is an enveloped, bullet-shaped, non-segmented, negative-strand RNA virus (NSRV) belonging to the rhabdovirus family, which includes the rabies virus. Because some attenuated VSV strains are nontoxic to normal tissue, VSV has therapeutic potential as an anticancer agent and vaccine vector (1, 2). Furthermore, pseudotypes of VSV carrying receptors for HIV proteins can selectively target and kill HIV-1–infected cells and control HIV-1 infection (3, 4).

Whereas many other NSRVs are pleomorphic, VSV has a rigid “bullet” shape. Attempts to visualize its organization by means of negatively stained electron microscopy (EM) have resulted in limited two-dimensional (2D) pictures: The virion has a lipid envelope (decorated with G spikes) that encloses a nucleocapsid composed of RNA plus nucleoprotein N and an associated matrix formed by M proteins. In recent years, crystal structures of components of VSV have been determined: the C-terminal core domain MCTD of the matrix protein (M) (5, 6), the nucleoprotein (N) (7), the partial structure of phosphoprotein (P) (8), the complex of N with the C-terminal of P (9), and the two forms of the ectodomain of the glycoprotein (G) (10, 11). The large polymerase (L) still awaits structure determination. However, how these proteins assemble into the characteristic rigid bullet-shaped virion has not been clear. Here, we report the 3D structure of the helical portion (the “trunk”) of the VSV virion as determined by means of cryo-electron microscopy (cryo-EM) as well as a study of the bullet-shaped tip from an integrated image-processing approach. This analysis leads us to propose a model for assembly of the virus with its origin at the bullet tip.

Cryo-EM images of purified VSV virions show intact bullet-shaped particles almost devoid of truncated or defective interference particles (Fig. 1A). The reconstructed map of the virion trunk (Fig. 1B and movies S1 and S2) has an effective resolution of 10.6 Å based on the 0.5 Fourier shell correlation coefficient criterion (fig. S2). We were able to dock crystal structures of the C-terminal domain of M [Protein Data Bank (PDB)1LG7 or 2W2R] (5, 6) and all of N (PDB 2GIC) (7) into our cryo-EM density map. The matching of several high-density regions in the cryo-EM map with α helices in the docked atomic models (Fig. 2A, M, and Fig. 3A, N) supports the validity of the cryo-EM map. These dockings also establish the chirality of the structure.

Fig. 1

Cryo-EM of VSV virion and 3D reconstruction of its helical trunk. (A) A typical cryo-EM micrograph of VSV virions at 98,000× magnification. The trunk portion is marked by the boxes. (Inset) Incoherent average of Fourier transforms of all raw images showing the layer lines. (B) Density map of the virion trunk. To enhance visual clarity and to show the interior, we computationally removed four turns of M, part of the membrane bilayer, incomplete subunits, and a 30° wedge. Nucleocapsid N and matrix M layers were displayed at a threshold of 1.15 σ above the mean; envelope densities were displayed at a threshold of 0.1 σ above the mean. (C) A complete repeat of the N and M helices, featuring 75 helical asymmetric units in two turns. (D) The central, vertical slice (17.3 Å thick) in the density map. (E) A radially color-coded surface representation of a central slab (23 Å thick). 1 and 2 indicate the outer and inner leaflets of the phospholipid bilayer envelope, respectively; t indicates the putative cytoplasmic tail of G; M is the matrix protein; N is the nucleoprotein. (Inset) Maps in all panels are colored according to radial distance as depicted in the scale bar. N,red to green; M,light blue to blue; envelope membrane, purple to pink. In this and Figs. 2 to 4, the arrow in every panel denotes the directionality (tip to trunk) of virions along the axis of the helix or of parts as they would be in the virion.

Fig. 2

In situ structure of the full-length M matrix protein. (A) Fit of the crystal structure (ribbon) of the C-terminal core domain MCTD (right) of M into the corresponding density map (mesh, contoured at 1.15 σ above the mean), taken from the cryo-EM map. The α helices are shown in red and β sheets are in purple. The numbered yellow spheres in the left part of the density map mark the positions of the four contact points on the M-hub domain. The highest density regions of the cryo-EM density are shown as gray-shaded surfaces by contouring at 3.0 σ above the mean. (B) (Left) Four adjacent M (light blue) with two N (green) subunits in the neighborhood of one M with its M-hub in yellow. The contact points on the M-hub that mediate interactions with M and N are labeled 1 to 4. The volume is contoured at 1.0 σ. (Right) By turning the left panel 80° around the vertical axis and removing the frontal M subunit, the interaction between N and M is illustrated.

Fig. 3

Formation of the bullet tip of VSV virion by the nucleocapsid (N) ribbon. (A) Fitting of the crystal structure (7) of nucleoprotein (N) (yellow ribbon) and RNA (blue ribbon) into the cryo-EM density map (semitransparent green, displayed at a threshold of 1.5 σ above the mean) from the VSV virion trunk. The helical axis in this panel points toward the reader. The purple wire frames represents the highest-density regions of the cryo-EM structure (threshold of 3.5 σ above the mean), which colocalize with α helices and the vRNA of the crystal structure. (Insets) Along the upper part of the interface between adjacent C lobes in the decamer, there are six hydrogen bonds (including R309 to E419) and one (I237:Y324) hydrophobic interaction (top right inset). After flexible docking of the atomic structure from the decamer into the cryo-EM density map of the trunk, distances between amino acid partners in these seven sites increase by ~9 Å, disrupting these interactions. (B) Comparison of the inclination of N subunits (green) in our cryo-EM structure from the trunk of the virion (37.5 subunits/turn) with the inclination of the N subunits (red) in the crystal structure from the decamer ring (10 subunits/turn) (7). (Top) Dashed lines through a side view of an N subunit from the trunk (left) and with an N subunit from the decamer (right) show the difference in tilt, the angle up from the horizontal plane. (Bottom) Dashed lines through end-on views of N subunits show the difference in dihedral angle between adjacent N subunits in the trunk (green) and in the decamer ring (red). (C) A representative class-average of the virion tip from 75 individual images. Numbers inside the nucleocapsid designate the order of N subunits in the nucleocapsid ribbon, which may be traced by following the path 1 > 1a > 2 > 2a, etc. (D) Negative-stain EM images of the wild-type decamer and two mutant rings confirm the importance of two of the interactions specified above. Both mutants produce rings larger than a decamer. (E) An illustration of a plausible process by which the nucleocapsid ribbon generates the virion head, starting with its bullet tip. The curling of the nucleocapsid ribbon generates a decamer-like turn at the beginning, similar to the crystal structure. When assembly nearly completes this turn, continuation of vRNA requires that the ribbon form a larger turn below it, similar to that in the mutants in (D). As the spiral enlarges and progresses to the helical trunk, the tilt of individual N subunits decreases. When it reaches the seventh turn, the nucleocapsid ribbon becomes helical (insets), in which each new turn of the nucleocapsid fits naturally under the preceding turn (insets).

When reconstructed with a helical symmetry imposed (12), the 3D map includes an outermost lipid bilayer, a middle layer composed of a helix-based mesh of M, and an innermost condensed nucleocapsid composed of a helically organized string of N subunits and RNA (Fig. 1, B and C, and fig. S3). [The M (Fig. 2A) and N (Fig. 3A) layers were identified on the basis of their close match with their crystal structures of M (5, 6) and N (7).] Each of the N and M layers consists of a single helix (1-start helix). Although the interior of the nucleocapsid contains a denser region than background, no density was found that was reasonably attributable to individual P or L subunits, suggesting that those are organized in a lower-symmetric or asymmetric fashion [supporting online material (SOM) text]. A layer of density outside the membrane may be identified as G protein (fig. S1) because the G protein is the only envelope protein in VSV. However, individual G spikes cannot be resolved in this layer, possibly because of flexibly attached ectodomains or inconsistent symmetry, and this layer of density was subsequently removed from the 3D reconstructions (12).

The outer diameter of the outer leaflet of the lipid bilayer is 700 Å. The exact length of the virion varies at 1960 ± 80 Å. The conical end comprises ~25% of the total length, and the cylindrical (helical) trunk comprises ~75%. The conical end contains approximately seven turns of a spiral before reaching a cylindrical (helical) trunk. The trunk of a typical virion contains ~29 turns. Each turn contains exactly 37.5 asymmetric units and rises 50.8 Å along the helical axis; two turns form a helical repeat (Fig. 1C). This value, 37.5, is very close to that estimated previously from scanning transmission EM [38 subunits per turn (13)] and from the N crystal structure [38.5 subunits per turn (7)]. Below, we also suggest that this value is consistent with geometric constraints on placement of G-trimers.

Docking of the crystal structure of N and RNA into the cryo-EM structure of the nucleocapsid also allows us to establish the directionality of viral RNA (vRNA) in the virion. The docked crystal structure shows that the 3′ end is at the conical tip of the bullet and the 5′ end is at the base of the trunk. We therefore follow this convention: The bullet tip defines the origin, and the helical trunk is “downward” from the origin. Arrows in Figs. 1 to 4 follow this convention.

Fig. 4

Architecture of the VSV virion. (A) Representative 2D averages of conical tip, trunk, and base of VSV and a montage model of the tip and the cryo-EM map of the trunk. N is green, M is blue, and the inner (2) and outer (1) leaflets of the membrane are purple and pink, respectively. (Inset) Illustration of the base region of the VSV virion. The “X” marks the absence of a turn of M helix below the lowest turn of the N helix. (B) Cryo-EM structure showing the putative cytoplasmic tail of G protein binding to an M subunit through a thin linker. The inner leaflet (2) of the membrane has unusual bumps (arrowhead) that meet M at the site of a thin linker density (arrow). (C) A wedge of the virion trunk, illustrating its geometric arrangement across the three layers. The N layer, the M layer, and the two membrane leaflets (1 and 2) are arranged in coaxial cylinders with their radii determined from our cryo-EM structure. Because of the difference in radii, the helical lattice points on the three layers form different triangles. The smaller the radius, the narrower the apex angle (inset). At the outer surface of the membrane, the lattice points form an equilateral triangle.

The full-length M protein in the cryo-EM structure of the virion trunk has two domains. The C-terminal domain (MCTD) was solved by means of x-ray crystallography (6), and we show the crystal structure fitted as ribbons in Fig. 2A. (The cross-correlation coefficient between the cryo-EM map and the fitted MCTD structure blurred to 11 Å is 0.63.) The α helices of the MCTD crystal structure colocalize with the highest-density regions in the cryo-EM map. The N-terminal domain (M-hub), which was not resolved in the crystal structures (5, 6), points toward the N layer (SOM text and fig. S5). The volume of the M-hub is consistent with its amino acid sequence length of 57 residues.

The M-hub contains four contact sites (Fig. 2, A and B, labeled 1 to 4, and Fig. 2B, yellow). M-hub contact point 1 connects the M-hub to an N subunit from the upper helical turn, whereas M-hub contact point 2 connects the M-hub to an N subunit from the lower helical turn. (By our convention, “upper” and “lower” indicate toward and away from the conical tip, respectively.) These dual vertical interactions set the M helix in an interleaved position between N helix turns and should provide stability and rigidity for the nucleocapsid as well as the M helix in the virion. These observations are consistent with results from mutagenesis, which show that amino acids 4 to 21 (in the M-hub) are important for nucleocapsid binding and viral assembly (14).

M-hub contact point 3 binds laterally to MCTD of the trailing M subunit from the same helical turn, whereas contact point 4 binds to the tip of the MCTD domain of the M subunit from an upper helical turn. The binding of M-hub contact point 4 sets the vertical spacing of adjacent turns of the M helix and by extension that of the N helix. It can be regarded as a “frame” that holds the N helix. It also leads to the formation of the 2D triangularly packed array (or “mesh”) within the outer (M) helix.

The protein-protein interactions revealed in our structure agree well with previous studies. For example, the surface of MCTD complementary to contact point 4 includes amino acids 120 to 128 (Fig. 2A, light blue arrow). Proteolytically opening this segment disrupts interactions between trypsin-cleaved M (Mt) and full-length M (5, 15, 16). Also, in a recent structural study of full-length M most of the M-hub is disordered and therefore poorly resolved, but M-hub contact point 4 (amino acids 41 to 52) along with its binding partner (MCTD residues 120 to 128) are well resolved (6), which is consistent with our finding that M-hub contact point 4 and the loop participate in binding. In addition, the self-association of M appears to follow nucleated polymerization, the “nucleus” consisting of three to four M subunits (17). Here, we suggest that these subunits join with each other at contact site 3.

N subunits encapsidate and sequester the genomic vRNA and form a higher-order linear structure in the shape of a ribbon through intermolecular interactions (fig. S3) (18). In our cryo-EM structure, the nucleocapsid is present as a helical tube with an inner radius of 154 Å and an outer radius of 225 Å (fig. S3). Individual N proteins within the helically coiled ribbon (Fig. 3B, green subunits) tilt upward by 27° from the horizontal plane (the plane perpendicular to the helical axis). Constrained by the 752 screw axis, one N subunit sits below and exactly in the gap between two other subunits from the turn above it (Fig. 1B and fig. S3). As seen from the volume representation (fig. S3), the turns of N are not densely packed against one another in the vertical direction. Thus, in the absence of the overlying M helix, the formation of a rigid nucleocapsid core would be impossible.

The crystal structure of an individual N subunit and its associated RNA has been solved for a ring of 10 N subunits: a “decamer ring” (7). This crystal structure can be unambiguously fitted into our cryo-EM density map. (The cross-correlation coefficient between the cryo-EM map and the final fitted N structure blurred to 11 Å is 0.70.) Each N subunit can be divided into an N-terminal lobe (Fig. 3A, top, N lobe) and a C-terminal lobe (Fig. 3A, bottom, C lobe) (movie S3). The N lobe points radially away from the helix axis and interacts with M proteins at its outer surface (Fig. 3A, top). As revealed in the crystal structure of the N decamer ring, RNA threads in a groove between the N lobe and the C lobe (Fig. 3A and movie S3). The density that connects adjacent N lobes is their bound vRNA. The cryo-EM structure of the C lobe in the trunk also agrees well with its crystal structure in the decamer. It has a more globular shape than the N lobe and faces the inner cavity (Fig. 3A, downward). In contrast to N lobes, C lobes bind to one another and therefore establish the lateral interactions in the inner (N) helix.

All rhabdoviruses have a bullet-shaped architecture. The 2D classification of cryo-EM images of bullet tips shows that these conical parts are identical among images (Fig. 3C and fig. S1). In all class averages, the nucleocapsid begins with fewer subunits per turn at the tip, progressing downward to the helical form in approximately seven turns. How does the nucleocapsid complete this structural arrangement? When the N crystal structure of the decamer ring is fitted into the trunk portion of a virion with 37.5 subunits per turn, the interface between the C lobes of adjacent N subunits must open markedly because of the larger dihedral angle between adjacent N subunits (Fig. 3B, bottom left versus bottom right). As a result, the six hydrogen bonds (including Glu419/Arg309) and the one hydrophobic interaction (Tyr324 to a pocket composed of Ile237 and part of Arg309) observed in the decamer are pulled apart by ~9 Å in the trunk portion of the virion (Fig. 3A, insets). These seven interactions might be used to achieve different energy modes for the formation of N rings of different sizes. Our mutagenesis studies support this hypothesis: Mutating either Arg309 or Tyr324 to Ala results in a preference for rings larger than the decamer observed in the wild type (Fig. 3D, table S1, and SOM text).

We propose that assembly starts at the apex of the virion tip, which is consistent with earlier suggestions (19). The nucleocapsid forms the sharp tip by using the first mode, as found in the decamer ring. Indeed, as shown in the 2D class averages, the first turn of the nucleocapsid matches the shape and size of projection images of the decamer ring (Fig. 3C and fig. S1). As it finishes one turn, the continuation of the vRNA strand forces the nucleocapsid from the first mode to one with a larger diameter, fewer interactions, and higher free energy. Indeed, mutating R309 or Y324 to alanine (artificially breaking one of the above seven interactions) appears to reduce the probability of the smallest ring size and promote the formation of larger rings (Fig. 3D). At the same time, stacking the second turn of the nucleocapsid onto the preceding turn permits binding of M. [Our 2D class averages of the base region of the virion show that the lowest turn of M lies between the lowest two turns of N (Fig. 4A, inset), suggesting that each M subunit must bind simultaneously to two N subunits.] A similar scenario continues until the nucleocapsid reaches its last mode, the mode that can repeat helically and contains a constant number of 37.5 subunits per turn in the trunk. This mode in turn might be stabilized by new interactions.

The M helix forms a triangularly packed lattice of M subunits (Figs. 1B and 4A). Next to the outer surface of the M helix, envelope membrane density intrudes inward at sites centered on each M subunit. There, a thin linker density runs from the membrane to contact an M subunit (Fig. 4B). This density is probably the cytoplasmic tail of a G protein, which is known to interact with M and is critical for budding (20, 21). Presumably, the G protein trimer binds three underlying M simultaneously (SOM text). If the trimer has threefold symmetry, the binding slots for G on the outside of the envelope membrane need to be an equilateral triangle. Indeed, we find that these sides are 58.6, 59.2, and 58.0 Å at the outer surface of the membrane. This geometric arrangement requires that the lateral spacing of M and N subunits within the M and N helices, each with progressively smaller diameters from G to M to N layers, be smaller and smaller. Our measurement of apex angles confirms that requirement (Fig. 4C). Given the base and height dimensions, the radial spans of N and M proteins, and the 27° tilt from the horizontal plane, these apex angles are satisfied only with 37.5 subunits per turn.

Assembly of a virus particle is generally presumed to be a stochastic process. However, assembly of VSV appears to follow a well-orchestrated program. It begins with RNA and N as a nucleocapsid ribbon (Fig. 3E). The ribbon curls into a tight ring and then is physically forced to curl into larger rings that eventually tile the helical trunk (Fig. 3E). M subunits bind on the outside of the nucleocapsid, rigidify the bullet tip and then the trunk, and create a triangularly packed platform for binding G trimers and envelope membrane, all in a coherent operation during budding.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S6

Table S1


Movies S1 to S6

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank S. Qiu and Xin Zhang for their technical assistance, Xiaorui Zhang for assistance in graphics, and M. Yang for proofreading the manuscript. This work was supported in part by NIH grants AI050066 (to M.L.), GM071940 (to Z.H.Z.), and AI069015 (to Z.H.Z.). P.G. was supported in part by a training fellowship from the W. M. Keck Foundation to the Gulf Coast Consortia through the Keck Center for Virus Imaging. The first octant of the helical reconstruction map of the virion trunk and its key segmentations have been deposited with the Electron Microscopy Data Bank (accession code EMD-1663). Flexibly fitted coordinates of N into our cryo-EM density have been deposited with PDB (accession code 2WYY).
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