X-ray crystal structure of the rotavirus inner capsid particle at 3.8 A resolution

Abstract

The rotavirus inner capsid particle, known as the "double-layered particle" (DLP), is the "payload" delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter approximately 700 A) in the asymmetric unit of the P2(1)2(1)2(1) unit cell of dimensions a=740 A, b=1198 A, and c=1345 A. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 A resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 A). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two "subdomain" boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: lambda1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a "5-fold hub" that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5'-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.

Fig 1

Cut-away view of the complete rotavirus virion (TLP), based on cryoEM reconstructions and assignments of densities. The two outer-layer proteins are in yellow (VP7) and red (VP5*/VP8*). The middle-layer protein (VP6) is in green; the inner-layer VP2, in blue. Coils of dsRNA (not shown here) fill the interior, organized in part by internal structures of the ICP described in this paper.

Fig 2

Stucture of VP2. A. Ribbon representation, with secondary structural elements labeled. Helices in dark blue; strands in light blue. B. Amino-acid sequence (single-letter code) of UK Bovine VP2 (serogroup A) with secondary structural elements shown above the sequence. C. The VP2 shell, viewed from outside the particle into the 5-fold channel. The central decamer is highlighted, with VP2A in dark blue and VP2B in light blue. D. Internal view of the VP2 shell, represented as a surface rendering with colors for VP2A and B as in panel C. Residues 81-100 (the last parts of the N-terminal arms) are represented, respectively, by white and gray “worms”.

Fig 3

Conformational flexibility of VP2 and VP2-like proteins: rotavirus VP2 (this work), BTV VP3 and reovirus λ1 . In each panel, there are orthogonal views of the monomer closer to the 5-fold (e.g., VP2A), in red and dark blue, superposed on the monomer farther from the 5-fold (e.g., VP2B), in green and light blue. The proteins were aligned on their central domains. Rotavirus VP2 and BTV VP3 each have similar relative shifts of their dimer-forming domains (lower part of each figure; rmsd 2.4 Å and 4.7 Å, respectively), but substantially different shifts of their apical domains (upper part of each figure; 2.1 Å and 7.9 Å, respectively). λ1 has only two domains, with an rmsd of 5.4 Å of one when the other is aligned.

Fig 4

The five-fold channel formed by VP2A monomers (backbone ribbons), with key conserved positively charged residues (Lys526, Arg527 and Arg531) shown as ball-and-stick representations. The VP2 loops (residues 354 to 363) that form an internal gate are highlighted in red.

Fig 5

Structure and packing of VP6. A. Ribbon representation . B. The VP6 shell: 260 VP6 trimers are colored relative to their positions with respect to the icosahedral symmetry axes: gold (T, on the 3-folds), red (T′, adjacent to T), dark blue (D, closest to the 2-folds), light blue (P, closest to the 5-folds), and purple (P′, adjacent to P). The inner VP2 layer is shown in red and green coils.

Fig 6

Alternative juxtapositions of VP6 trimers across local twofold axes in the T=13 icosahedral lattice. The interfaces between T and P′ and T and D (blue) show a relative shift of ~15 Å relative to those between T and T and T and T′ (tan). T-T has been aligned to T-D. Despite this shift, similar residues interact at the trimer interface, generating a set of alternative salt bridges, as described in the text.

Fig 7

Interactions of VP6 and VP2. A. View of the VP2 layer directly along the icosahedral 3-fold, highlighting (in green) residues that have direct contacts with the VP6 trimer bound over them. A hydrophobic pocket on the surface of VP2 (side chains shown) receives a loop from VP6 (residues 65 to 72). B. Detailed view of this loop, which can adopt any of several conformations, depending on the part of the VP2 surface it contacts; it is represented here in the conformation it adopts when interacting with the hydrophobic pocket shown in A.

Fig 8

View of the ICP surface, colored by B-factor (low to high, green to red, respectively). The degree of disorder increases with radial position in the particle and with distance within the surface from the nearest icosahedral 3-fold axis.

Fig 9

Internal features of the electron density map and their interpretation. A. Contoured map in the vicinity of a fivefold axis, showing layers of density (magenta) attributed to coiled RNA. Scale shows radius (distance from center) in Å. B. The fivefold hub. Density from a 2Fo-Fc map, filtered to 7 Å resolution and contoured at 0.9σ, in the region around the fivefold axis boxed in A. C. Schematic diagram of the interior structures. The relative spatial relationship of these features is shown with suggested connections (dotted lines) indicating that the N-terminal extension of VP2 may form an internal channel.

Fig. 10

Schematic cross-section of rotavirus interior, illustrating dsRNA packing. The diagram is approximately to scale (for an internal radius of ~240 Å for the VP2 shell and for D, the packing diameter of the dsRNA segments, ~ 33Å). The dsRNA should be pictured as coiled around each fivefold hub, with approximate hexagonal close packing in cross section. The principal density of the RNA helices is about 20 Å in diameter; the lighter region around each cross-section shows the hydration layer.

Fig. 11

Model for DLP assembly, VP2 is in blue (dark and light for A and B conformers, as in Fig. 2); VP1, in red; VP3, in orange; RNA segments, in magenta. VP1 recruits (+)-RNA and VP3, and these components come together with five VP2 dimers to form the fundamental assembly unit. Twelve such units (one lacking RNA and potentially lacking VP1 and VP3) come together to form a DLP; synthesis of (−)-RNA inside the particle reels in the (+)-strand. The mechanism by which one of each RNA segment is incorporated remains unspecified.