X-ray structure of a native calicivirus: structural insights into antigenic diversity and host specificity

Abstract

Caliciviruses, grouped into four genera, are important human and veterinary pathogens with a potential for zoonosis. In these viruses, capsid-related functions such as assembly, antigenicity, and receptor interactions are predominantly encoded in a single protein that forms an icosahedral capsid. Understanding of the immunologic functions and pathogenesis of human caliciviruses in the Norovirus and Sapovirus genera is hampered by the lack of a cell culture system or animal models. Much of our understanding of these viruses, including the structure, has depended on recombinant capsids. Here we report the atomic structure of a native calicivirus from the Vesivirus genus that exhibits a broad host range possibly including humans and map immunological function onto a calicivirus structure. The vesivirus structure, despite a similar architectural design as seen in the recombinant norovirus capsid, exhibits novel features and indicates how the unique modular organization of the capsid protein with interdomain flexibility, similar to an antibody structure with a hinge and an elbow, integrates capsid-related functions and facilitates strain diversity in caliciviruses. The internally located N-terminal arm participates in a novel network of interactions through domain swapping to assist the assembly of the shell domain into an icosahedral scaffold, from which the protruding domain emanates. Neutralization epitopes localize to three hypervariable loops in the distal portion of the protruding domain surrounding a region that exhibits host-specific conservation. These observations suggest a mechanism for antigenic diversity and host specificity in caliciviruses and provide a structural framework for vaccine development.

Fig. 1.

X-ray structure of SMSV. (A) A sample region in the electron density map with modeled amino acid residues 175–189 (APAPTALATLATAST). (B) The x-ray structure of SMSV viewed along the icosahedral twofold axis (capsid protein subunits are shown as Cα trace). Locations of a set of A/B and C/C dimers and icosahedral fivefold and threefold axes are denoted. The NTA (internal, not visible in this view), S domain, and P1 and P2 subdomains of the subunits are colored in green, blue, yellow, and orange, respectively. (C) A ribbon representation of the B subunit structure. The NTA (residues 163–200, green), S domain (residues 201–361, blue), and P1 (residues 362–413 and 590–703, yellow) and P2 (residues 414–589, orange) subdomains are indicated. BIDG and CHEF β-strands in the S domain are denoted. The arrowhead indicates the location of the Pro-178; solid and dashed arrows indicate the location of the hinge (residues 359–361) between the S and P domains and the location of a strictly conserved Gly-414 at the P1/P2 junction, respectively. The sequence–structure relations for SMSV (along with that in rNV for comparison) are shown in Fig. 5, which is published as supporting information on the PNAS web site.

Fig. 2.

The NTAs of SMSV form a novel network of interactions. (A) The network of interactions among NTAs of subunits A, B, and C (colored in blue, red, and green, respectively) in the SMSV capsid. The icosahedral fivefold, threefold, and twofold axes are denoted. (B) A view from inside of the capsid showing domain-swapping interactions between the twofold-related subunits (A’B, CC’, and AB’) and hydrophobic interactions at the local threefold axis between the α-helices from the subunits belonging to neighboring asymmetric units. The icosahedral and the local threefold axis are labeled as 5, 3, 2, and 3’, respectively. The S domains of the A, B, and C subunits (slate, orange, and yellow, respectively) in an icosahedral asymmetric unit and their dimer-related subunits (B’, A’, and C’ in red, blue, and green) are shown as surface representations, whereas their respective NTAs in the same colors are shown as ribbons.

Fig. 3.

Dimer interactions and interdomain orientations. The dimeric interactions in the A/B dimers of SMSV (A) and rNV (B) structures (Protein Data Bank ID code 1IHM). Ribbon representations of the AB subunits involved in the dimeric interactions are shown within a semitransparent surface of the dimer; in each case the P1 subdomain in the A subunit is highlighted in green, whereas the rest of the structure is shown in blue, and the B subunit in each case is shown in red. (C) Stereo view of the superposition of the SMSV (magenta) and rNV (green) subunit structures, indicating significant changes in the interdomain orientations between the two structures.

Fig. 4.

Structure of the P2 subdomain and mapping of the FCV neutralizations sites. Ribbon representations (Upper) of the P2 subdomains in SMSV (A) and rNV (B) along with their respective topology diagrams (Lower). The β-strands are labeled from A’ to F’ in each case. The loops containing the FCV neutralization epitopes are indicated for SMSV. (C Upper) Surface representation of the A/B dimer as viewed from outside the capsid (approximately along the dimeric twofold axis) showing the N-terminal (NT) HVR, central conserved region, and C-terminal (CT) HVR in red, blue, and cyan, respectively. (C Lower) Sequence comparison of the representative sequences from SMSV (top four sequences corresponding to SMSV4, SMSV5, primate, and SMSV1), canine calicivirus, and FCV (bottom three sequences corresponding to FCV6, FCV4, and FCV9) in the conserved region flanked by the NT and CT HVRs showing host-dependent conservation (see also Fig. 6).