Non-enveloped virus particles (those that lack a lipid-bilayer membrane) must breach the membrane of a target host cell to gain access to its cytoplasm. So far, the molecular mechanism of this membrane penetration step has resisted structural analysis. The spike protein VP4 is a principal component in the entry apparatus of rotavirus, a non-enveloped virus that causes gastroenteritis and kills 440,000 children each year1. Trypsin cleavage of VP4 primes the virus for entry by triggering a rearrangement that rigidifies the VP4 spikes2. We have determined the crystal structure, at 3.2 Å resolution, of the main part of VP4 that projects from the virion. The crystal structure reveals a coiled-coil stabilized trimer. Comparison of this structure with the two-fold clustered VP4 spikes in a ∼12 Å resolution image reconstruction from electron cryomicroscopy of trypsin-primed virions shows that VP4 also undergoes a second rearrangement, in which the oligomer reorganizes and each subunit folds back on itself, translocating a potential membrane-interaction peptide from one end of the spike to the other. This rearrangement resembles the conformational transitions of membrane fusion proteins of enveloped viruses3,4,5,6.
VP4 spikes on virions have ‘head’, ‘body’, ‘stalk’ and ‘foot’ regions (Fig. 1a), formed by the two VP4 trypsin cleavage fragments VP8* and VP5* (Fig. 1b). The VP8* fragment contains a globular domain, the ‘VP8* core’, which forms the head7. In some virus strains the VP8* core is a haemagglutination domain, which mediates initial cell attachment by binding sialic acid. Portions of both VP8* and VP5* make up the body. VP5* residues in the body are implicated in membrane penetration and integrin binding8,9,10. The carboxy-terminal part of VP5* forms the foot, which is buried beneath the VP7 layer, anchoring the spike. Neutralizing antibodies against rotavirus bind VP8*, VP5* and VP7 and block cell entry11. When present in the gut lumen, they protect against rotavirus gastroenteritis12. Thus, knowledge of the structural basis for cell entry can inform vaccine development.
The VP5* fragment used for structure determination (VP5CT; Fig. 1b) is produced by serial chymotrypsin and trypsin cleavage of a VP4 precursor13. Expressed directly, the fragment is insoluble and probably misfolded. VP5CT has the authentic VP5* amino terminus at A248. VP4 cleavage to produce this N terminus is required to prime particles for cell entry and membrane interaction14,15. The crystal structure of VP5CT contains rhesus rotavirus (RRV) VP4 residues N252–L523. Perfect hemihedral twinning prevented use of diffraction data beyond 3.2 Å resolution in the structure determination (see Methods and Supplementary Methods).
VP5CT is a well-ordered homotrimer that resembles a folded umbrella (Fig. 2a). The ‘post’ of the umbrella is a C-terminal, α-helical, triple coiled-coil. Each of the three panels comprising the ‘shade’ of the umbrella is an N-terminal globular domain (Fig. 2b). Each globular domain packs in a groove between the α-helices of the other two subunits on the post's negatively charged outer surface (Fig. 2c). Just above the post, strands K, H, and G from each globular domain join in a continuously hydrogen-bonded β-annulus around the three-fold axis (Fig. 2a). At the top of the structure, propeller-like packing of tryptophan side chains (W262, strand B) creates an additional trimer contact. Between the β-annulus and the W262 propeller, a cavity (volume 390 Å3) centred on the three-fold axis leaves room for potential rearrangements. In full-length VP5*, the foot region (Fig. 1a), similar in mass to VP5CT, is attached to the coiled-coil.
The core of each globular domain is an eight-stranded anti-parallel β-sandwich (Fig. 2b, light and dark blue). Two features of potential functional importance project from its top edge: the GH and CD β-hairpins. By joining with strands K in the β-annulus, the GH hairpin clamps each globular domain in the ‘folded umbrella’ position. The flexible tip of the CD β-hairpin bears a sequence motif (DGE) implicated in rotavirus binding to α2β1 integrins (Fig. 2b, e)10. The accessibility of the motif, which protrudes into solvent, supports the proposed receptor-binding function.
The tips of loops projecting from the bottom edge of the β-sandwich impart a hydrophobic apex to the globular domain (Fig. 2d), which may function in membrane penetration. Near the β-sandwich, portions of these loops form β-sheets F′G(H/H′) and B′C′E′D′ (purple and pink respectively in Fig. 2b). The unusual interposition of β-strand I (green) between these sheets creates a deep ‘hydrophobic bowl’. The distal part of the F′G loop and the alphavirus ‘fusion loop’ have similar sequences8. Both loops lie at the hydrophobic tips of globular domains implicated in membrane disruption, but the domains and the loops themselves are otherwise structurally dissimilar (Supplementary Fig. 1 and Tables 2, 3)6. Thus, the sequence similarity probably reflects selection for hydrophobicity and flexibility in both loops, but not common ancestry. Each strand of the F′G loop has an in-register glycine–glycine pair (382–383 and 399–400, located under labels F′ and G in Fig. 2b). The four conserved glycines might provide a hinge, opening up the hydrophobic bowl beneath and allowing relatively extensive and flexible membrane interactions during cell entry.
How does the VP5CT structure relate to that of the VP4 spike? The region of VP5CT from Y267 to L470 neatly fits the molecular envelope of the spike body in a new, ∼12 Å resolution electron cryomicroscopy image reconstruction of trypsin-primed virions (Fig. 3b, e). As Y267 and L470 hydrogen bond to each other on adjacent β-strands B and J, this region constitutes a self-contained globular domain called the VP5* antigen domain (solid colours in Fig. 3c, f). VP5CT residues outside the antigen domain must fold differently in the spike than in the trimeric crystal structure (white outlines in Fig. 3c, f).
Together, the VP5* antigen domain and the VP8* core contain all known neutralizing epitopes on VP4 (Fig. 3a, d), making them logical minimal antigens for recombinant rotavirus vaccines. The much greater conservation in VP5* suggests stronger functional constraints and correlates with heterotypic neutralization by some VP5*-specific antibodies. The VP5* antigen domain is thus a particularly promising potential immunogen. On the basis of the VP5CT structure, we have grouped VP5* neutralization escape mutations into five epitopes (Supplementary Table 1). The epitopes are solvent-exposed on both the VP5CT trimer (Fig. 2c, d) and the spike body (Fig. 3a, d). The surfaces of the trimer that lack neutralization epitopes are inaccessible on the spike, explaining their antigenic ‘silence’ on primed virions.
Most VP5*-specific neutralizing monoclonal antibodies map to epitope 5-1, the exposed surface of the ‘shoulder’ at the virion-distal end of the spike body, formed by the hydrophobic apex of the antigen domain (Fig. 3). The head covers much of the apex, but part of the F′G loop is exposed. Monoclonal antibody 2G4 selects a neutralization escape mutation in this loop at residue Q393 (ref. 8). Electron cryomicroscopy of antibody-binding fragment (FAb)-decorated virions shows that 2G4 indeed binds the shoulder16, confirming our fit of the antigen domain to the spike body. Antibody 2G4 blocks a post-attachment entry event11, which is possibly domain rotation or membrane insertion (see below). Epitopes 5-1, 5-2, 5-3 and 5-4 are linked by a network of antibody competition and escape mutant cross-resistance (references in Supplementary Table 1). Epitope 5-5 is outside of this network and is mimicked by a short synthetic peptide17. It maps to the flexible CD hairpin, adjacent to the integrin-binding motif (Fig. 2b, c, e), suggesting that epitope 5-5-specific antibodies may block integrin interactions.
Image reconstructions from electron cryomicroscopy of rotavirus particles have provided good evidence for two conformational states of VP4: an uncleaved, flexible state, which does not produce visible density in averaged image reconstructions2 (Fig. 4a), and a trypsin-cleaved, rigid state with approximate two-fold symmetry of the parts that project beyond the VP7 shell (Figs 3b, e and 4b)18,19. Contrary to the two-fold character of the projecting part of the spikes on trypsin-primed virions, the VP5CT crystal structure reveals an unambiguous trimer (Fig. 2a, c, d). The three-fold contacts bury 3,956 Å2 (25.8%) of the surface of each subunit and render the trimer resistant to dissociation by SDS13. Such extensive and stable contacts provide structural evidence that this region of VP4 evolved to trimerize. VP5CT thus represents a third conformational state for the projecting part of VP4 (Fig. 4c).
How can a two-fold to three-fold reorganization of VP4 occur? For the transition to take place on the virion, rather than after dissociation, each spike location must actually contain three VP4 subunits at all stages. Indeed, recently obtained image reconstructions of trypsinized rotavirus particles treated briefly at pH 11 reveal foreshortened spikes with clear three-fold character: trefoil-like features projecting slightly above the VP7 layer (B.V.V.P., manuscript in preparation). Unfolding at elevated pH appears to cause each VP4 subunit to condense so that better-ordered segments near the foot produce strong density in the image. We conclude that two-fold clustering after trypsin cleavage leaves the third subunit still flexible, as in the uncleaved state (Fig. 4b).
Several observations support this view of VP4 organization. The spike projects from an asymmetric, angled stalk, so that its approximate two-fold axis is displaced from the centre of the foot18,19. Thus, the symmetry of the foot and the projecting spike need not match. Indeed, image reconstructions of VP4 obtained by subtracting images of spikeless particles from images of particles with spikes reveal three-fold symmetry in the foot19. The symmetry mismatch between foot and projection was previously reconciled by proposing that two VP4 subunits each contribute three similarly shaped domains to the foot. The new data suggest, instead, that the three-fold symmetry of the foot reflects the true subunit stoichiometry. Our previous biochemical data on VP5CT oligomers in solution were consistent either with trimers or with tight dimers that associate weakly into tetramers (dimers of dimers)13. At the time, the latter interpretation appeared more consistent with electron microscopy, but the crystal structure and new image reconstructions resolve the ambiguity in favour of trimers.
If just two of three VP4 subunits at each spike location cluster after trypsin cleavage, interactions with VP7 and/or VP6 must select the two that form the visible spike. The VP8* and VP5* fragments of the third, flexible molecule need not remain associated, and this subunit could even perform a different function in cell entry. Interactions with VP7 and VP6 may also arrest the progression of VP4 rearrangements at the primed stage.
In the dimeric spike, a loop (F′G) with the properties of a membrane-interacting element points away from the foot, and the GH hairpin points approximately 90° away from the dyad axis of the body (Fig. 3b, c, e, f). In the VP5CT trimer, the F′G loop points towards the C terminus of the coiled-coil, to which the stalk and foot attach, and the GH hairpin joins the β-annulus around the three-fold axis (Fig. 2a). Therefore, in the two-fold to three-fold reorganization, the VP5* antigen domain must rotate approximately 180° about an axis roughly perpendicular to the spike's long axis (Fig. 4b, c). This rotation translocates the hydrophobic tip of the F′G loop by at least 55 Å from the shoulder of the spike towards the foot.
The two subunits of the spike body make proximal and distal dyad contacts, leaving a gap in the centre (Fig. 3b, e). The VP4 residues that participate in these contacts are outside the relatively rigid VP8* core and VP5* antigen domain and probably control their large-scale displacements. Both termini of the VP8* core are in the ‘neck’ between head and body, near the distal dyad contact. Both termini of the VP5* antigen domain are at the bottom of the body, adjacent to the proximal dyad contact. The antigen domain completely fills the electron microscopy envelope between the dyad contacts, leaving no unfilled density to suggest an unmodelled connecting strand. Therefore, VP8* residues outside the core (M1–V64) must form the distal dyad contact and tether head to body. During rotation of the VP5* antigen domain, disruption of the distal dyad contact would release VP8* (Fig. 4c). Similarly, VP5* residues outside the antigen domain (A248–Q266 and I471–L523 and perhaps additional C-terminal residues that connect to the stalk) must form the proximal dyad contact. In the trimeric state, these residues make up the elements of the three-fold contacts: β-sheet ABJ, β-strand K and the α-helix (Figs 2a and 3c, f). The refolding of residues in the proximal dyad contact to form the coiled-coil may drive the two-fold to three-fold reorganization.
The three distinct conformations of VP4 correspond to steps in rotavirus entry (Fig. 4a–d). When uncleaved virions are released from infected cells, three VP4 molecules with flexible stalks occupy each of 60 symmetry-equivalent positions (Fig. 4a). In the gut lumen, trypsin cleavage between VP8* and VP5* produces a first rearrangement, which primes VP5* for membrane attack (Fig. 4b). Dimeric interactions in the external portion of two primed VP4 subunits rigidify the spikes, elevating and presenting the VP8* core for cell-surface ligand binding. The third VP4 molecule remains flexible. The heads of the spike shield hydrophobic prominences on the body. We hypothesize that a subsequent, as yet unknown, entry-associated event triggers a transition to the trimeric conformation seen in the crystal structure (Fig. 4c). In this second transition, disruption of the dimer contacts releases VP8* from VP5* and unmasks the hydrophobic apex of the VP5* antigen domain, which may insert into a host-cell membrane. As the trimeric coiled-coil zips up, the antigen domain folds back against it. The resulting translocation of the potential membrane-interaction loop towards the foot could disrupt a cellular membrane. This disruption may in turn create a breach that allows a subviral particle to enter the cytoplasm, or it could lower the local calcium concentration, leading to virion uncoating and subsequent entry events. The fold-back transition could also alter interactions with receptors or directly trigger virion uncoating.
The essential properties of VP4—its capacity to adopt an inactive, precursor conformation; a primed, receptor-binding conformation; and a final, folded-back conformation—recall those of enveloped virus fusion proteins3,4,5,6. Understanding these transformations will allow us to analyse neutralization mechanisms, design subunit immunogens, and elucidate molecular details of membrane perforation.
Expression and purification of RRV VP5CT
VP5CT was produced by serial chymotrypsin and trypsin digestion of purified RRV VP4 from recombinant baculovirus-infected insect cells, as described previously13. Selenomethionine-substituted protein was produced by metabolic incorporation in insect cells20.
VP5CT crystallized at 23 °C in hanging drops containing equal volumes of well solutions and a protein solution containing 9 mg ml-1 VP5CT, 20 mM Tris, pH 8.0, 1 mM EDTA, 0.02% sodium azide and 0.1 mM benzamidine. Selenomethionine-substituted VP5CT crystals that formed after 4 days with a well solution of 6% ethanol, 50 mM Tris pH 7.0 and 1 mM dithiothreitol were harvested after one month and flash-frozen with liquid nitrogen in a freezing buffer of 11% ethanol, 50 mM Tris, pH 7.0, 17% 2-methyl-2,4-pentanediol (MPD) and 5 mM dithiothreitol. Unsubstituted VP5CT crystals that formed after 3 weeks with a well solution of 500 mM ammonium sulphate, 5.5–6% MPD and 50 mM PIPES pH 6.5 were harvested after 1–7 months and flash-frozen in 600 mM ammonium sulphate, 22.5% MPD and 20–50 mM Tris, pH 8.0.
Diffraction data were scaled in space group P4(2)22, with six monomers per asymmetric unit. Starting phases were determined by multiple wavelength anomalous dispersion, based on diffraction from crystals of selenomethionine-substituted VP5CT. Thirty seleniums per asymmetric unit were located by direct methods, using SnB21. Landmarks provided by seleniums and bulky side chains unambiguously aligned the primary amino acid sequence to the electron density. Refinement took advantage of six-fold non-crystallographic symmetry. The final model contains no Ramachandran violations, and 76.6% of dihedral angles are in most-favoured regions. Pseudocentring and perfect hemihedral twinning in VP5CT crystals has been described previously22. Details of the structure determination are provided in Supplementary Methods, Supplementary Table 4, and Supplementary Fig. 2.
Electron micrographs of rotavirus particles (strain SA11-4F) embedded in a thin layer of vitreous ice were recorded under low electron dose conditions (∼ 10 e- Å-2) at ×50,000 magnification on a JEOL 4000 electron microscope operated at 400 kV. Electron micrographs were digitized with a scanning interval corresponding to 2.8 Å in the object. A three-dimensional reconstruction was computed using cylindrical expansion methods23,24 to 12.4 Å resolution by combining 908 particles from 15 micrographs with appropriate corrections for contrast transfer function. Defocus values, determined using EMAN software25, ranged from 0.8 to 3-µm underfocus. As is conventional23, to compute initial maps, electron microscopy data were first analysed in reciprocal space, using only 5-2-2 symmetry. To compute the final map, full icosahedral symmetry was imposed by three-fold real-space averaging after confirming the presence of three-fold symmetry in initial maps. The protruding portion of the VP4 spike was computationally extracted from the final density map. Crystal structures were fit to the electron cryomicroscopy map by a combination of automated searching using Situs26 and manual fitting.
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We thank M. Babyonyshev for technical assistance; T. Yeates for help in analysing the crystal twinning disorder; H. Greenberg for cloned genes and recombinant baculoviruses; E. Vogan for help with data collection and analysis; and the staff of Advanced Photon Source beamline ID-19 (Argonne National Laboratory) and Cornell High Energy Synchrotron Source beamlines F1 and A1. We acknowledge the use of electron cryomicroscopy facilities at the National Center for Macromolecular Imaging funded by NIH at Baylor College of Medicine. This work was supported by an NIH grant and an Ellison Medical Foundation New Investigator in Global Infectious Diseases award to P.R.D., by an NIH grant to B.V.V.P., and by an NIH grant to S.C.H., who is a Howard Hughes Medical Institute Investigator.
The authors declare that they have no competing financial interests.
Comparison of the rotavirus VP4 F'G loop and the alphavirus E1 fusion loop. Includes an amino acid sequence comparison and a structural comparison. (PDF 158 kb)
Sample of electron density. An image from an unaveraged simulated annealing omit map is shown. (PDF 1474 kb)
Details of the crystallographic structure determination. Includes a description of the twinning disorder in the crystals. (DOC 35 kb)
Neutralization escape mutations. Mutations that map to the VP5* fragment are assigned to epitopes based on the VP5CT structure. (DOC 62 kb)
Rotavirus VP4 sequences used to assess variability and obtain a consensus sequence. The variability in these sequences is mapped onto the surfaces of the VP8* core and VP5* antigen domain in Fig. 3a, c. The consensus sequence is used to assess the similarity between the VP4 F'G loop and the alphavirus E1 fusion loop in Supplementary Fig. 1. (DOC 37 kb)
Alphavirus E1 sequences used to obtain a consensus. The consensus sequence is used to assess the similarity between the VP4 F'G loop and the alphavirus E1 fusion loop in Supplementary Fig. 1. (DOC 26 kb)
X-ray diffraction data collection, phasing, and refinement statistics. (DOC 47 kb)
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Dormitzer, P., Nason, E., Venkataram Prasad, B. et al. Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature 430, 1053–1058 (2004). https://doi.org/10.1038/nature02836
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