Fucose Binding Cancels out Mechanical Differences between Distinct Human Noroviruses

Figures

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  Figure 1. Representative images of tested noroVLPs of norovirus variants and the comparison of their sizes. (a) AFM images and (b) the corresponding height profiles of GI.1, GII.17, and GII.10 at pH 7.0. The height profiles were taken at the dashed lines of the respective colour in the corresponding images. The protruding outer layer and the inner layer were depicted by the contrast of light and dark on the surface of particles in AFM images and the protrusions and depressions on height profiles, respectively. The height scale of AFM images is 40 nm. (c) Scheme of the AFM setup for studying noroVLPs. The AFM cantilever with a sharp tip at its end was used for imaging and indenting an individual noroVLP immobilized on a hydrophobic glass cover slip. A laser beam is focused on the rear side of the cantilever to detect its deflection. (d) Scheme of noroVLPs whose P domain of VP1 is at resting (left) and rising (right) conformation. The flexible hinge region connecting a P domain and a S domain is depicted as an orange string. (e) The corrected height distribution of tested noroVLPs. The corrected heights of noroVLPs of each norovirus variant were acquired from three independent measurements.…

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  Figure 2. Native MS of different noroVLPs. The size distribution in 150 mM ammonium acetate at pH 7.4 at 20 µM is exemplarily shown for GI.1 Norwalk (pink), GII.17 Kawasaki (blue), and GII.10 Vietnam (green) from bottom to top. Ion distributions at approximately 23,000, 39,000, and 44,000 m/z were assigned to T = 1, T = 3, and putatively T = 4 complexes, respectively, according to the predicted charge by the Rayleigh model [39,40].

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  Figure 3. Typical approach lines of the F-D curves of the tested norovirus variants at pH 7.0. The viral spring constant of each linear phase was calculated by linear fitting at each linear phase (indicated by dashed lines in the graph). The critical force and corresponding critical indentation of each linear phase were measured from each discontinuous point (indicated by navy blue arrows in the graph) on the F-D curves. In the follow-up analysis, the first two linear fitting slopes and the ordinates and abscissas of the first two discontinuous points were included for the statistical analysis.

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  Figure 4. Fucose treatment diminished the original differences in the mechanical properties of the tested variants. The mechanical properties of noroVLPs at (a) pH 7.0 and (b) pH 7.0 after incubating with fucose were acquired from three independent measurements. The vertical column represents the mean, and the error bar represents the standard error of the mean. A one-way ANOVA test was used for significance analysis. p values are indicated by asterisks: p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), and not significant (N.S.).

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  Figure 5. Alkaline treatment affected the mechanical properties of GI.1 and GII.17, leading to a similar mechanical performance of the two variants. The mechanical properties of noroVLPs of GI.1 and GII.17 at pH 9.0 were acquired from three independent measurements. The vertical column represents the average, and the error bar represents the standard error of the mean. A one-way ANOVA test was used for significance analysis. No significant (N.S.) indicates the p value is larger than 0.05.

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  Figure 6. Mechanical model of (a) GI.1 and (b) GII.10 noroVLP deformation under the applied force. The rising conformation of the P domain of GII.10 capsid protein VP1 is depicted by the unfolded hinge connecting the P and S domains (indicated by the orange string in (b)). The navy blue arrows in the model indicate the tension pointing outward generated by the dimeric interactions between P domains. The dark green arrows indicate the inward force of the S domain acting on the P domain, thereby creating a balanced state of forces in the capsid. In the representative F-D curves of noroVLPs of GI.1 and GII.10, the corresponding events on particles when the first two discontinuous points happened are depicted and indicated by blue arrows.