Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM

Abstract

Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 A) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal "arms" of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca(2+)-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.

Fig. 1.

Rotavirus structure determined by cryo-EM. (A) Structure of the complete virion filtered at 25-Å resolution. The segmentation of the structure is based on reconstructions of the complete virion and the VP7-coated DLP and on published work of others (–23). (B) Cryo-EM image of VP7-coated DLPs (7RP). (C and D) Regions of the VP7 density (filtered to 4.2-Å resolution) corresponding to strands β2, β1, β3, and β11 and helix αB. (E) Domain organization of VP7 (17). Domain I (light yellow) is a Rossmann fold; domain II (dark yellow), a β-jelly roll. The N- and C-terminal extensions (gray) become ordered when VP7 associates with the DLP. Numbers below the bar are those of the first residue in each segment. Disulfide bonds are indicated as residue numbers joined by lines across the top of the bar. The signal peptide is 50 residues long. Numbering corresponds to rhesus rotavirus, serotype G3.

Fig. 2.

VP7 outer protein layer. (A) Structure of a VP6–VP7 heterohexamer, derived from crystal structures of VP6 (green) (14) and VP7 (gold) (16), docked into the cryo-EM density, and from the model built into cryo-EM density for the N-terminal arm. Two of the three VP7 N termini forming tight interactions with VP6 can be seen on the left and right side of the VP6 trimer. The red square indicates the area shown in more detail in B. (B) VP7 N terminus (residues 58–78) and corresponding cryo-EM density, showing its interaction with VP6. Pro-58 and Asn-69 are labeled; the latter bears a glycan for which density is present. Strands A′ and A″ of VP6 are also labeled. The density corresponding to residues 61–68 is weaker than in other parts of the map because this part of the structure lacks a clearly defined secondary structure and makes no contact with other parts of the structure. The map was filtered at 4.5-Å resolution to display a continuous density trace in this region. (C and D) Conformational differences between bound VP7 (gold) and the VP7 crystal structure (red) (16). The VP7 trimer is viewed from the outside of the virus along its symmetry axis (C) and from the “side”, normal to its symmetry axis (D). The way in which the domain hinge displacement “flattens” the subunit when the trimer binds VP6 is evident in the latter view. The alignment is based on a superposition of the Rossmann-fold domains.

Fig. 3.

Interface between 2 VP7 trimers. The interface is shown from the outside of the virus (A) and from the “side”, normal to its symmetry axes (B). The N-terminal arms between residues 58 and 78 are shown as red curves; the approximate positions of the arms between residues 51 (the N terminus) and 57 are shown as dotted green curves, illustrating that the N termini may contribute to intertrimer contacts.

Fig. 4.

Conformational differences between DLP and 7RP. The structures of VP2 and VP6 docked into the 7RP cryo-EM density are shown in green whereas the DLP structure (12) is shown in red. A narrowing of the central channel on the icosahedral 5-fold axis and an inward movement of the VP2 and VP6 layers in the 7RP, compared with the DLP, are the principal differences. The VP7 layer of the 7RP is not shown (for clarity); the green arrow indicates the “clamping down” of the VP6 and VP2 layers that accompanies VP7 binding.