Norovirus Escape from Broadly Neutralizing Antibodies Is Limited to Allostery-Like Mechanisms

FIG 1. Structure of MNV. The top stereo diagram shows the surface of the MNV T=3 capsid, with the A, B, and C subunits shown in blue, green, and red, respectively. The A′-B′ and E′-F′ loops are highlighted in cyan and wheat colors, respectively. The 3-fold and 5-fold axes are noted. The bottom stereo figure shows a side view of the A and B subunits. The P and S domains are noted and the A′-B′ and E′-F′ loops are noted as described above. Note how the P domains float above the S domain. Also shown are the open and closed conformations of the A′-B′/E′-F′ loops.

FIG 2. 5C4 monoclonal antibody (mAb) binds to noroviruses. (A) Neighbor-joining phylogenetic tree of 10 murine norovirus (MNV) strains using the 541-amino-acid capsid protein. (B and C) Plates were coated with MNV strains (B) or human norovirus (HuNoV) VLPs (C) and tested for reactivity to 1 μg per well of MAb 5C4. MNV1 and mock lysate were used as positive and negative controls, respectively. The dashed line represents twice the absorbance values at 415 nm (A415) for the negative control and indicates the threshold for a positive signal. OD, optical density. (D) MNV strains were incubated with the indicated concentrations of MAb 5C4 for 30 min at 37°C before a plaque neutralization assay was performed. Virus titers were determined after 48 h of incubation and are shown relative to the absence of antibody. Data are presented as means ± standard errors of the means (SEM) of the results from at least three independent experiments.

FIG 3. Cross-neutralizing activities of anti-MNV1 MAbs. (A to C) Plates were coated with equal amounts of MNV strains and tested for reactivity to 1 µg per well of MAbs A6.2 (A), 4F9 (B), and 2D3 (C). MNV1 and mock lysate were used as positive and negative controls, respectively. The dashed line represents twice the absorbance values at 415 nm (A415) for the negative control and indicates the threshold for a positive signal. (D to F) MNV strains were incubated with the indicated concentrations of MAbs A6.2 (D), 4F9 (E), and 2D3 (F) for 30 min at 37°C before a plaque neutralization assay was performed. Virus titers were determined after 48 h of incubation and are shown relative to the absence of antibody. Data are presented as means ± SEM of the results from at least three independent experiments.

FIG 4. Cryo-EM image reconstruction of the 2D3/MNV complex fitted with the atomic models of the MNV P domain dimer and two of the MAb 2D3 Fabs. (A) Surface rendering of the entire virus colored according to the particle radius. (B) Center section of the same structure. The Fab constant domains are shown in dark blue, the variable domain in cyan, and the P domain in yellow/orange. The shell domain is orange, and the RNA interior is purple. (C) A stereo image of the Situs fitted components corresponding to the cryo-EM density data. The heavy chains are shown in green, the light chains in yellow, and the two conformations of the P domain, A and B, in red and blue, respectively.

FIG 5. Comparison of the binding conformations of MAbs A6.2 and 2D3. (A) Stereo image of surface rendering of the two MNV complexes, with MAb 2D3 shown in yellow and MAb A6.2 in transparent blue. (B) Stereo image of the Situs fitted models with MAb A6.2 shown in blue and MAb 2D3 in red. The P domain is shown in green.

FIG 6. Comparison of the interactions between MAb 2D3 and the open and closed P domain conformations of MNV. The model shown represents the results of application of the molecular dynamics flexible fitting method (MDFF) (44) to the starting Situs model. The light and heavy chain variable domains of MAb 2D3 are shown in orange and green, respectively. The open and closed conformations of the P domain are shown in red and blue, respectively. Also noted are the locations for MAb 2D3 escape mutations V339I and D348E (shown with mauve and yellow spheres, respectively). Note that the CDR3 of the Fab loop fits well between the A′-B′ and E′-F′ loops of the open conformation of the P domain but overlaps with the A′-B′ loop in the closed conformation.

FIG 7. Comparison of the MAb A6.2 and MAb 2D3 antibody contact areas. To calculate the contact areas, the refined Fab/MNV models were analyzed using PDBePISA (46) for just the Fab contacts with the open conformation. The surface areas of the open and closed conformations are shown in red and blue, respectively. The residues in the open conformation that contact the bound antibodies are shown in wheat color. Also noted are the locations for the MAb A6.2 escape mutations on the E′-F′ loop.

FIG 8. Stereo images of the environments around the MAb 2D3 escape mutants in the two P domain conformations. (Left) The V339I mutation in the context of the open conformation, with the modeled mutation shown in green and the possible residues affected by the mutation highlighted in yellow. (Right) In contrast to the open conformation, there is a significantly more space around I337 in the closed conformation that could accommodate the additional methyl group.

FIG 9. Stereo image of the local environment of the D348E mutation. The modeled mutation is shown in green, and possible contacts are highlighted in yellow. This mutation lies on a loop that is on the side of the P domain and not near the bound 2D3 antibody.

FIG 10. Stereo image of the effects of the V339I escape mutation on the in silico molecular dynamics of the 2D3 Fab/MNV complex. The variable regions of the heavy and light chains are shown in green and orange, respectively, and the open and closed conformations in red and blue, respectively. The minimized wild-type structure is shown in the darker hues, while the structures seen after the molecular dynamics experiments are shown in lighter hues. While the minimized wild-type structure remained relatively stable (RMS deviation of ~1 Å), the V339I structure showed significant long-range disruptions (RMS deviation of ~2.5 Å). The salt bridges affected by the mutation are shown as cyan spheres labeled with the names of the amino acid pairs. The V339I and D348E escape mutations are denoted by mauve and yellow spheres, respectively.