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, 83 (4), 1754-66

Rotavirus Architecture at Subnanometer Resolution

Affiliations

Rotavirus Architecture at Subnanometer Resolution

Zongli Li et al. J Virol.

Abstract

Rotavirus, a nonturreted member of the Reoviridae, is the causative agent of severe infantile diarrhea. The double-stranded RNA genome encodes six structural proteins that make up the triple-layer particle. X-ray crystallography has elucidated the structure of one of these capsid proteins, VP6, and two domains from VP4, the spike protein. Complementing this work, electron cryomicroscopy (cryoEM) has provided relatively low-resolution structures for the triple-layer capsid in several biochemical states. However, a complete, high-resolution structural model of rotavirus remains unresolved. Combining new structural analysis techniques with the subnanometer-resolution cryoEM structure of rotavirus, we now provide a more detailed structural model for the major capsid proteins and their interactions within the triple-layer particle. Through a series of intersubunit interactions, the spike protein (VP4) adopts a dimeric appearance above the capsid surface, while forming a trimeric base anchored inside one of the three types of aqueous channels between VP7 and VP6 capsid layers. While the trimeric base suggests the presence of three VP4 molecules in one spike, only hints of the third molecule are observed above the capsid surface. Beyond their interactions with VP4, the interactions between VP6 and VP7 subunits could also be readily identified. In the innermost T=1 layer composed of VP2, visualization of the secondary structure elements allowed us to identify the polypeptide fold for VP2 and examine the complex network of interactions between this layer and the T=13 VP6 layer. This integrated structural approach has resulted in a relatively high-resolution structural model for the complete, infectious structure of rotavirus, as well as revealing the subtle nuances required for maintaining interactions in such a large macromolecular assembly.

Figures

FIG. 1.
FIG. 1.
Structure of mature rotavirus particle. (A) A representative region from a micrograph is shown. Individual particles (indicated in red circles) were extracted and processed. (B) The resulting 9.5-Å resolution reconstruction is shown. The capsid layers are radially colored such that red represents VP4 spikes, yellow is the VP7 layer, blue is the VP6 layer, green is the VP2 layer, and orange is the internal density (RNA and polymerase complex).
FIG. 2.
FIG. 2.
Structure of the VP4 spike. (A) A VP4 spike segmented from the cryoEM map is shown. The domains of VP4 (VP8*, VP5*-t, bridge, and base domains) are indicated on the left. The asterisk indicates a breakpoint between the base and the bridge domains. An arrow indicates the bridging domain shown in Fig. 6. (B) Fitting of VP8* crystal structure (PDB ID: 1KQR) into the cryoEM density map shown in two orientations. A clear depression corresponding to the sialic acid binding site is visualized in the VP8* domain (denoted by a circle and shown in the inset image). The sialic acid molecule can be seen extending out of the density. (C) Fitting of the individual monomers from the VP5*-td crystal structure into the spike density. (D) Comparison of the fitting of the VP5*-td dimer as a whole (in cyan), VP5*-tt (in yellow; two of the three subunits in the trimer are shown), and the monomers from VP5*-td (red).
FIG. 3.
FIG. 3.
Connecting VP8* and VP5*-t. (A) Fitted structures of VP8* and VP5*-t are shown in one of the dimeric subunits of the VP4 spike. The polypeptide chain is colored from the N (blue) to C (red) terminus. A difference map corresponding to the density unaccounted for by the VP8* and VP5-t crystal structures is shown in yellow. (B) The amino acid sequence of the linker region from residue 225 to 249 with the predicted β-strand in blue is shown. (C) This sequence was putatively modeled in the context of the difference map, shown in green (arrow in panel A and as a stereo view in panel C. This linker interacts with the other VP5*-t subunit (in gray) in the VP4 spike. (D) The interface between the VP8* and VP5*-t domains is shown in two views.
FIG. 4.
FIG. 4.
Base domain of VP4. (A) The density corresponding to the base domain, which sits inside the type II channel, is shown. The base domain becomes visible at a radius of 305 Å and terminates at a radial distance of ∼375 Å. (B) Serial sections of the base domain, along its length from bottom to top (vertical line in panel A). The slices are contoured from high density (blue) to low density (red). The threefold correlation coefficient is also indicated. (C) Plot of threefold correlation coefficient over the entire base domain.
FIG. 5.
FIG. 5.
Secondary structure elements in the base domain. Secondary structure elements, as detected using SSEHunter (2), are shown. Helices are indicated as green cylinders, while β-sheets are shown as cyan polygons. A clear threefold pattern is again evident.
FIG. 6.
FIG. 6.
Bridging domain of VP4. (A) Fitting of the helix bundle from the trimeric VP5*-tt crystal structure to the bridging domain between the base and VP5*-t is shown. The helix bundle was fit both as a dimer (left) and as a trimer (right). While not unambiguous, the dimer appears to fit the density better. An arrow and an asterisk indicate density features illustrated in Fig. 2.
FIG. 7.
FIG. 7.
VP4 model. (A) A side view of the type II channel shows that the base of VP4 is clearly wider than the surface opening in the VP7 layer. However, the trimeric VP4 base appears to rest in the VP6 layer. Interactions of VP4 with VP6 and VP7 can be seen. (B) Application of the bilateral filter revealed the presence of additional density (denoted by a red circle) that is contiguous with a small protrusion emanating from the base domain (indicated by an asterisk in Fig. 2A and 7A). This density could represent a portion of the possible third flexible VP4 subunit. The third subunit protrudes outward from the top of the base domain.
FIG. 8.
FIG. 8.
Interactions between VP7 and VP6 layers. (A) Interacting VP7 and VP6 trimers, colored in yellow and blue, respectively, are shown in top and side views. (B) The crystal structure of VP6, shown in the left panel, was fit to the segmented T=7 layer subunit, revealing the boundaries between VP6 and VP7. Comparison of the SSEHunter results in VP6 was used to assess the accuracy of secondary structure identification in this region. In the right panel, the secondary structure elements identified with SSEHunter compare favorably to those from the X-ray structure. (C) The contact regions between VP6 and VP7 are shown in two views. Two interaction points between VP6 and VP7 subunits are indicated by arrows. (D) Secondary structure elements in VP7 identified using SSEHunter are shown along with the VP6 crystal structure. SSEHunter identified three helices, which are labeled H1, H2, and H4; numbering is based on the sequence-based predicted secondary structure described in “T=13 capsid layers” in the text.
FIG. 9.
FIG. 9.
VP4 interactions with the VP7 and VP6. VP5* makes contacts with three of the six VP7 trimers, at a point indicated by an asterisk in Fig. 8B, surrounding the type II channel. (A) One interaction is between the C-terminal residues of VP5*-t and a VP7 trimer (indicated by the asterisk). (B) The other two interactions, with two other VP7 trimers, occur at the end of the bridging domain (indicated by the number sign and asterisk). (C and D) VP4 also contacts the VP6 layer through its base domain. (C) Only three of the six VP6 trimers, indicated by asterisks, surrounding the type II channel, show interactions with the base domain. (D) A side view of the type II channel depicting the VP4 interactions with both VP7 and VP6. (E) A zoomed-in view showing the points of interaction between VP4 and the fitted VP6 crystal structure. The VP6 X-ray structure is colored in magenta, corresponding to the color scheme in panel A; regions of the VP6 X-ray structure that contact VP4 are colored in gray.
FIG. 10.
FIG. 10.
T=1 VP2 layer. (A) VP2A (pink) and VP2B (light green) subunits in the icosahedral asymmetric unit are shown. The VP6 trimers that sit atop VP2 subunits are indicated by triangles. The VP6 residues, as deduced from fitting of the VP6 crystal structure, that interact with VP2 are shown. (B) The SSEHunter results are shown for both VP2A and VP2B subunits. (C) Comparison of the secondary structure elements to the structural homologue in bluetongue virus (VP3, right) allowed for the assignment of the VP2 sequence to the visualized helices (Table 1). (D) Helix 0, indicated with a red circle, protrudes toward the fivefold vertex. However, which of the VP2 subunits that helix 0 is associated with could not be determined. (E) Zoomed-in view of the interactions between VP6 residues and the VP2 layer.

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