Conformational Flexibility in Capsids Encoded by the Caliciviridae

Abstract

Caliciviruses are a diverse group of non-enveloped, positive-sense RNA viruses with a wide range of hosts and transmission routes. Norovirus is the most well-known member of the Caliciviridae; the acute gastroenteritis caused by human norovirus (HuNoV), for example, frequently results in closures of hospital wards and schools during the winter months. One area of calicivirus biology that has gained increasing attention over the past decade is the conformational flexibility exhibited by the protruding (P) domains of the major capsid protein VP1. This was observed in structure analyses of capsids encoded by many species and is often a consequence of environmental cues such as metal ions, changes to pH, or receptor/co-factor engagement. This review summarises the current understanding of P-domain flexibility, discussing the role this region plays in caliciviral infection and immune evasion, and highlighting potential avenues for further investigation.

Keywords: P-domain; calicivirus; capsid; cryo-EM; structure.

Figure 2

Amino acid sequence alignment of highly variable antigenic regions A−H from selected HuNoV GII.3 and GII.4 human norovirus isolates, located on the major capsid protein VP1. Residue locations (from Tohma et al. and Lo et al.) are illustrated by boxes and brackets. Antigenic region A: residues 294, 295, 296, 297, 298, 368, 372, 373. B: 333, 382. C: 339, 340, 341, 375, 376, 377, 378. D: 393, 394, 395, 396, 397. E: 407, 411, 412, 413, 414. F; 327, 404. G: 352, 355, 356, 357, 359, 364. H: 309, 310 [51,54]. Residues are coloured according to biochemical properties. Pink = non-polar (Gly, Ala, Ser, Thr); orange = hydrophobic (Cys, Val, Ile, Leu, Pro, Phe, Tyr, Met, Trp); green = polar (Asn, Gln, His); blue = negatively charged (Asp, Glu); Red = positively charged (Lys, Arg). ORF2 sequences were downloaded from GenBank [55] and aligned and visualised in Jalview 2.11.4.0 [56]. GenBank sequence identifiers and protein IDs can be found in Supplementary Table S1.

Figure 3

(A) Simplified representation of P-domain rotation and collapse. Image created in Biorender. (B) Cartoon diagram of suggested loop movements in the resting and rising P-domain positions in the P2 regions of an A/B dimer. A dimer is coloured cyan, B dimer is coloured purple. G’H’ loop is coloured red, C’D’ loop is coloured orange, E’F’ loop is coloured light orange, and A’B’ loop is coloured yellow. Note the significant upwards movement of the C’D’ loop in the rising P-domain position. Adapted from [66]. PDB of resting position: 7N6Y [67]. PDB of rising position: 7L5J [68].

Figure 1

(A) Simplified phylogenetic illustration of the Caliciviridae adapted from the phylogenetic tree presented in the ICTV Virus Taxonomy Profile [1]. The tree is based on amino acid sequences of the major capsid protein VP1. The tree illustrates the 11 recognised genera (Sapovirus, Bavovirus, Nacovirus, Vesivirus, Nebovirus, Lagovirus, Recovirus, Valovirus, Norovirus, Minovirus, and Salovirus) and 13 recognised species of the Caliciviridae alongside illustrations of representative hosts (non-exhaustive). (B) Cryo-EM structure of FCV [22], coloured according to the radius (key) with three protomers coloured to identify the positions of the A (purple), B (cream), and C (magenta) quasiequivalent positions of VP1. Depicted below the cryo-EM structure are schematic representations of the HuNoV, FCV, and MNV genomes. Adapted from [4]. The 5′ end of calicivirus genomes is covalently linked to viral protein genome-linked (VPg) proteins and a poly(A) tail of variable length at the 3′ end (depicted as p(A)). ORF1 encodes a viral polyprotein that is cleaved by virally encoded proteases into multiple non-structural proteins. In all three viruses, ORF2 and ORF3 encode the major capsid protein VP1 and minor capsid protein VP2, respectively; both are translated from a bicistronic subgenomic RNA (sgRNA) at later stages of infection. FCV ORF2 also encodes a leader of the capsid (LC) protein. ORF4, found only in MNV, is a fourth overlapping reading frame within ORF2 that encodes virulence factor 1 (VF1). NTPase = Nucleoside triphosphatase, Pro = Protease, RdRp = RNA-dependent RNA polymerase, N-term = N-terminal protein, Hel = Helicase. (C) Ribbon diagram illustrating the N-terminal domain (blue), shell domain (cyan), P1 (red), and P2 (yellow) domains of the major capsid protein VP1 (PDB 6GSH [22]).

Figure 4

Simplified schematic of GCDCA binding locations on HuNoV (green) and MNV (red) P-domains. Image created in Biorender. GCDCA binds MNV in a hydrophobic binding pocket, with each P-domain binding two bile acid molecules [79]. Conversely, GCDCA appears to bind HuNoV in a bile acid binding pocket at the apical sides of P-domains, being held by four to six residues (depending on strain) with a network of hydrophobic interactions and each P-domain binding a single bile acid molecule.

Figure 5

Cryo-EM maps of different preparations of TuV show either (A) well-defined, resting pose P-domains, or (B) poorly resolved C/C-dimers (purple arrows), that are shown to be in a raised and tilted pose by focussed classification (white arrow). Atomic models of C/C-dimers in resting (C) and raised (D) states show how the P-dimer tilts and rotates, by unfolding of the S-P linker at amino acid residues Pro203-Pro211 (pink arrow).