Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 86 (12), 6470-80

Epitope Insertion at the N-terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T = 3 Capsid Protein Leads to Larger T = 4 Capsids

Affiliations

Epitope Insertion at the N-terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T = 3 Capsid Protein Leads to Larger T = 4 Capsids

Daniel Luque et al. J Virol.

Abstract

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

Figures

Fig 1
Fig 1
Biochemical and structural analysis of VP1 insertion mutants. (A) Scheme of wt and chimeric VP1 proteins used, indicating length (right). Inserts are indicated. N42 and L42 have the same inserted sequence. The C42 insert sequence is shown in Fig. 6. Acidic residues, red; basic residues, blue; polar residues, green; hydrophobic residues, black. (B) Coomassie blue-stained SDS-PAGE gels of wt and chimeric VP1 assemblies used for cryo-EM data acquisition. Molecular size markers (MWM; ×10−3 Da) are on the left. (C) Cryo-EM of wt VP1 (top), L17 (middle), and N42 (bottom) capsids. Arrows indicate capsids larger than normal T=3 capsids. Scale bar, 100 nm.
Fig 2
Fig 2
Assessment of the resolution of wt and chimeric VP1 capsids. (A) FSC resolution curves were calculated for wt (blue), L17 (purple), L42 (orange), N42 T=3 (green), N42 T=4 (dashed green), and C42 (brown) capsids. The resolutions at which the correlations dropped below 0.5 and 0.3 are indicated. For the 0.5 threshold, the values for wt, L17, L42, N42 T=3, N42 T=4, and C42 capsid were 10.3, 9.9, 10.3, 20.0, 24.4, and 14.7 Å, respectively; values for the 0.3 threshold were 8.8, 8.6, 8.8, 18.6, 23.2, and 12.0 Å, respectively. (B) FSC resolution curves were calculated for the merged data set (wt, L17, and L42 capsids), with P domain spikes (S+Pwt+L17+L42, green) or without (Swt+L17+L42, purple), and compared to the wt VP1 capsid (blue). For the 0.5 threshold, the values for the merged data set with spikes or without were 8.9 and 8.1 Å, respectively, and for the 0.3 threshold the values were 7.8 and 6.9 Å, respectively.
Fig 3
Fig 3
Three-dimensional cryo-EM of wt VP1 capsid. (A) Surface-shaded representations of the T=3 capsid outer (left) and inner (right) surfaces viewed along an icosahedral 2-fold axis. The positions of two VP1 dimers, A/B and C/C, are indicated. Icosahedral symmetry axes are numbered. Plug-like densities at the 3-fold axis on the inner surface are in red. Six triangle-shaped densities around a 3-fold axis are marked (blue circles; the inset shows a magnified view). Maps are contoured at 2 σ above the mean density (the transparent surface is contoured at 1 σ). (B) RHDV VP1 secondary structural elements (SSE). Segmented dimers A/B (left) and C/C (right) with their SSE: α-helices (red cylinders) and β-sheets (blue planks). Black arrows indicate the α-helix at the basement of the S domain that contributes to the triangle-shaped structures (and corresponds to the NTA α-helix); red arrows indicate the α-helix in the S domain. Note that two additional α-helices are identified at the P1 subdomain in C subunits but not in A or B subunits. To emphasize that the interactions occur only in P2 subdomains, dimers (contoured at 3 σ) are viewed from a different orientation than that in Fig. 5D and F. Inside views are also shown (bottom).
Fig 4
Fig 4
Multiple-sequence alignment and homology model of VP1. Multiple-sequence alignment of VP1, SMSV, and FCV CP amino acid sequences. Identical residues, white on red background; partially conserved residues, red. VP1 SSE reflects the consensus of several SSE predictions (see Materials and Methods), and those of SMSV (2gh8) and FCV (3m8l) CP are assigned based on their crystal structures. Color coding for SMSV CP is as initially shown by Prasad's group (9); N-terminal arm, green; S domain, blue; P1 subdomain, yellow; P2 subdomain, orange. The FCV CP X-ray structure was obtained from strain FCV-5 (GenBank accession no. DQ910790). Our study was performed with FCV Urbana strain (GenBank no. NC_001481). The two sequences have 90.42% identity and are the same size. The thick red line indicates the region equivalent to the N-terminal hypervariable loop used in this study (GSGNDITTANQYDAADIIRN).
Fig 5
Fig 5
Quasiatomic model of RHDV T=3 capsid. (A) Complete T=3 capsid (white) with the VP1 quasiatomic model docked. The two VP1 dimer types are indicated (A/B, blue/red; C/C, green/green); white triangles define two icosahedral asymmetric units (ABC). Subunits with subscripts are related to A, B, and C by icosahedral symmetry (e.g., B to B5 by a 5-fold rotation). The locations of 5-fold, 3-fold, and 2-fold axes are indicated. (B) A 50-Å-thick RHDV VLP slab viewed along an icosahedral 3-fold axis. P2 subdomain-mediated interactions can be seen in some A/B and C/C dimer sections. (C) A quarter of a 50-Å-thick RHDV VLP slab viewed along an icosahedral 2-fold axis. VP1 domains are color coded (P2, orange; P1, yellow; S, blue; NTA, green). Red densities indicate 2 of the 20 different densities at the 3-fold axis on the capsid inner surface, which were calculated by subtracting the capsid quasiatomic model from the wt cryo-EM capsid. (D and E) A/B contact, viewed along the line joining the 3- and 5-fold axes (D) and from the inside (E). Bent contact between S domains (D) and molecular swapping of NTA domains (E) are shown. (F and G) C/C contact, viewed along the line joining two 3-fold axes (F) or from inside (G). Planar contact between S domains (F) and molecular swapping of NTA domains (G) are shown. Icosahedral symmetry axes, black symbols. Diagrams of bent and planar contacts and the NTA exchanges are shown.
Fig 6
Fig 6
VP1 molecular switch. (A) T=3 VP1 capsid viewed down a 3-fold axis from inside, showing NTA domains. N termini of B subunits (red) end at the 5-fold axis; A and C N termini converge at the 3-fold axis into a conspicuous unoccupied density. The last visible N-terminal residue is indicated by a sphere. Black symbols indicate icosahedral symmetry axes (asterisks indicate local 3-fold axes in which three α-helices interact). (B) View as described for panel A, with accessible inner surfaces represented with electrostatic potentials, showing the distribution of negative (red) and positive (blue) charges. Hexagons mark the absent densities at the 3-fold axis in the pseudoatomic T=3 capsid. Note the negative grooves defined among the NTA segments.
Fig 7
Fig 7
C42 capsid structure. (A) Surface-shaded representation of the C42 T=3 capsid outer surface viewed along an icosahedral 2-fold axis. Additional protruding densities, corresponding partially to the inserted epitope at the 3- and 5-fold axes, are red. Bottom, inserted sequence at the VP1 C terminus is shown (FMDV B and T epitopes, gray- and yellow-shaded regions, respectively). (B and C) Difference map calculated by subtracting wt VP1 from C42 capsid. The resulting difference map is shown in red on the outer surface of a C42 capsid viewed along icosahedral 3-fold (B) and 5-fold (C) axes. Fitted pseudoatomic models of the A, B, and C subunits of VP1 are color coded as described for Fig. 2 (A, blue; B, red; C, green). C-terminal ends are indicated by spheres.
Fig 8
Fig 8
NT42 T=3 and T=4 capsid structures. (A) Surface-shaded representations of the outer (top) an inner (bottom) surfaces of the NT42 T=3 capsid, viewed along an icosahedral 2-fold axis at 20-Å resolution. A, B, and C subunits are shown as ribbons. Inner surfaces of NT42 (bottom left) and wt (bottom right) T=3 capsid show the differences at the 3-fold axis (arrows indicate the sectioned 3-fold axis). (B) Outer (top) and inner (bottom) surfaces of the NT42 T=4 capsid, viewed along an icosahedral 2-fold axis at 25-Å resolution. The icosahedral asymmetric unit is shown (A, blue; B, red; C, green; D, yellow). (C) S domains in the T=4 (top) and T=3 (bottom) icosahedral shells. Interacting surfaces between A, B, C, and D β-barrels are quasiequivalent. (D) Images of T=4 (row 1) and T=3 (row 4) capsids taken directly from original cryomicrographs compared to the projected views (T=4, row 2; T=3, row 3) of the 3DR in the corresponding orientation. Selected capsids are oriented close to a 2-fold (column 1), 3-fold (column 2), and 5-fold (column 3) symmetry axis.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

MeSH terms

Associated data

Feedback