Unraveling the molecular basis of membrane-associated release of coxsackievirus B3

Figures

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  Fig. 1. Structural analysis of coxsackievirus B3 protomer (PDB: 1COV) highlighting the residue N63 and the possible interactions involved with Y173. All the images were generated using the PyMOL Molecular Graphics System, Version 3.0.5 Schrödinger, LLC. (A) Cartoon representation of the viral protomer showing VP1 (orange), VP2 (red), VP3 (blue), and VP4 (yellow). The inset focuses on residue N63 of VP3 and its proximity to Y173 of VP2. (B) Interactions of amino acids between and with aromatic rings in CVB3 protomer. Blue residues are involved in Pi-stacking interactions (yellow dashed lines), while green residues are involved in Pi-cation interactions (red dashed lines).

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  Fig. 2. Amino acid conservation at position 63 of VP3 protein across the genus Enterovirus. The bar chart represents the percentage of sequences with specific amino acids at this position for different species within the genus Enterovirus. Conservation values for asparagine (N) are expressed as a percentage, with the corresponding number of sequences analyzed indicated within each bar.

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  Fig. 3. Membrane-associated release of N63 mutants. Boxplot showing the percentage of viral progeny released as detergent-sensitive high-weight complexes, (bP/(bP + S). See text for details. Statistically significant differences between WT and mutants are indicated with black lines and symbols, while the differences between N63L and other mutants are indicated with blue symbols. *P < 0.05, **P < 0.01, ****P < 0.001 (one-way ANOVA with Tukey´s multiple comparison test).

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  Fig. 4. Effect of N63 mutations on viral spread. (A) Growth curves of the non-mutated CVB3 (WT) and mutants N63H, N63Y, N63F, N63W, N63D, and N63L. Viral spread was monitored by measuring mCherry fluorescence at various time points post-inoculation. (B) Violin plot comparing mCherry signal at 21 hpi for WT and N63 mutants. Significance levels are represented as follows: ****P < 0.0001 (one-way ANOVA with Tukey´s multiple comparison test). Significant differences between WT and mutants are indicated with black lines and symbols, while the differences between N63L and other viruses are indicated with blue symbols. (C) Representative images showing the progression of infection in cells infected with WT and N63 mutants at 21 hpi.

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  Fig. 5. Plaque phenotypes of N63 mutants in overlays of agar and agarose at 48 hpi. (A) Images of plaque assays performed with agar and agarose. (B) Boxplot comparing the plaque sizes (in mm²) for each virus under each condition. The red boxes represent plaque sizes on agar, while the blue boxes represent plaque sizes on agarose. ****P < 0.0001 (one-way ANOVA with Tukey´s multiple comparison test). (C) Heat map showing the ratio of plaque sizes on agarose to those on agar for each virus. (D) Heat map comparing plaque sizes on agar relative to WT virus. (E) Heat map comparing plaque sizes on agarose relative to WT virus.

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  Fig. 6. Molecular dynamics simulations of CVB3 WT virus and mutants at VP3 residue N63. (A) RMSD of the capsid over 5 µs of the simulation. (B) Radius of gyration (Rg) over the time of the simulation. (C) RMSF per residue of the viral polyprotein. (D) Violin plot of SASA for VP2-Y173 (pink), and position 63 of VP3 (gray). (E) Percentage of native contacts (Cons%) and accuracy (Acc%) at the first and last microseconds of the simulation.