The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation

Figures

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  Fig 1. Temporal and phylogenetic distribution of S:A222V. (a) Global phylogeny of 11,166 sequences belonging to G clade. The red inner circle denotes sequences with S:A222V, the external circle indicates PANGO linages of interest indicated in legend. (b) Percentage of global sequences with S:A222V (blue area) in different VOCs. Sequences belonging to parental lineages designated as B.1.177 appear as a green line, B.1.1.7 as a yellow line, B.1.617.2 as a red line, AY.4.2 as a purple line, and B.1.1.529 as a blue line. (c) The ratio of nonsynonymous to synonymous substitution rates (dN/dS) for the codon 222 of the spike for each period with a predominant variant, the size of the circle indicates–log(p) with a constant value of 10. *dN/dS for B.1.1.529 period is even higher but cannot be represented because dS = 0.

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  Fig 2. Temporal and phylogenetic distribution of the S:A222V in B.1.617.2 sequences. (a) Percentage of sequences designated as parental B.1.617.2 and its sub-lineages (designated as AY) with S:A222V by month. (b) Maximum likelihood phylogeny of 10,049 sequences from Lineages B.1.617.2 and derivatives. Phylogeny rooted with reference MN908947.3. Red external ring indicates sequences with S:A222V, the colour of branches indicates B.1.617.2 sub-lineages of interest. The scale bar indicates the number of nucleotide substitutions per site. Each circle in branches correlates with the bootstrap value; only bootstraps from 70 to 100 are represented.

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  Fig 3. S:A222V does not alter the neutralization capacity of convalescent sera. The sensitivity to neutralization by convalescent sera of pseudotyped VSV carrying the ancestral Wuhan-Hu-1 (Wuhan), S:D614G, or the [S:A222V + S:D614G] (20E) spike protein was evaluated. No significant differences were detected between any of the sera (n = 6; p>0.05 by Kruskal-Wallis test; p>0.05 by Mann-Whitney test for Wuhan-Hu-1 vs. S:D614G, Wuhan-Hu-1 vs. 20E, or S:D614G vs. Wuhan-Hu-1).

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  Fig 4. Cryo-EM of the [S:A222V + S:D614G] mutant. (a) Side view of the cryo-EM density map of the [S:A222V + S:D614G] mutant obtained after consensus of three independent runs of symmetry relaxation 3D classification, followed by initial model generation, refinement without symmetry imposition and deepEMhancer map sharpening, shown at 3 σ. Bar = 30 Å. (b) Fourier Shell Correlation (FSC) resolution curve (top) shown as the regular cryoSPARC global FSC resolution output, which includes no mask and different masks. Resolution based on the gold standard 0.143 criterion is 3.4 Å. The angular distribution coverage profile is also shown (bottom). (c) Atomic model of the [S:A222V + S:D614G] mutant shown as ribbon diagrams. S protein subunits are coloured in blue (chain A), red (chain B) and green (chain C). Glycan molecules are shown as stick diagrams and coloured according to their corresponding subunits.

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  Fig 5. Effects of the S:A222V mutation on the structure of SARS-CoV-2 spike. (a) On the left, cartoon representation of the trimeric spike. On the right, detail of the region of interaction between the NTD from subunit B (salmon) and the CTD1 region from subunit A (blue). The side chains of residues surrounding A222 (upper panel) or V222 (lower panel) are shown as sticks. (b) Comparison of the structures for the S:D614G and [S:A222V + S:D614G] mutants showing main conformational changes observed when S:A222V is present. Detailed view of the trimeric spike surface shown in semi-transparent representation with different colours for the different subunits, and RBDB, RBDA and NTDB domains from the S:D614G and [S:A222V + S:D614G] mutant structures shown as cartoon. Domains corresponding to the S:D614G structure are coloured in yellow. Arrows indicate the direction and magnitude of the observed domain movements. A dashed line represents the distance between the Cα atoms of residues 114 and 381 in chain B, which is ~ 3 Å higher in S:D614G than in [S:A222V + S:D614G]. (c) Schematic representation of the RBD and NTD from different subunits to show the movements observed when comparing the two…

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  Fig 6. PCA for the three MD simulated systems. (a) PCA projection of the first two high-variance motions for the S:D614G (in red) and [S:A222V + S:D614G] (in blue) mutants of SARS-CoV-2 S1 subunit of the glycan-free spike protein. (b) Conformational evolution of the NTD and RBD along the first eigenvector (PC1) mainly accounting for the RBD closed-to-open transition. The open and closed states at the two ends of the PC1 are highlighted in orange and green, respectively. (c) Conformational evolution of the NTD and RBD along the second eigenvector (PC2) accounting for lateral RBD+NTD fluctuations. An orange gradient is applied here to distinguish between different lateral orientations of the open NTD-RBD pair at its two ends. (d) Continuous population densities along the PC1 for a PCA relative to the RBD (residues 330–530; PC1RBD): solid profiles are used for the simulated closed, 3-down and open, 1-up states from the glycan-free systems based on 6VXX and 6VSB while continuous and dashed lines are used to define population densities for the simulated open 1-up states of the cryo-EM and fully-glycosylated S:D614G (red) and [S:A222V + S:D614G] (blue) mutants.

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  Fig 7. Dynamic network analysis for a subsystem consisting of the open RBD and its neighbouring NTD. (a) Open RBD of the simulated 1-up (UDD) SARS-CoV-2 Spike mutants. (b) Residue communities (3 for NTD, in teal, and 4 for RBD, in yellow) engaged in inter-domain contacts, along with their position in the protein sequence. The domains shown here are those highlighted with gray transparency in (a). (c) Communication pathways are highlighted with connecting lines whose thickness is proportional to the inter-domain betweenness centrality values. Orange connections represent the S:D614G and violet the [S:A222V + S:D614G] mutant; data is reported for the three systems: glycan-free from 6VSB (UDD), glycan-free from our cryo-EM data (cryo-UDD), and glycosylated from 6VSB (gly-UDD).

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  Fig 8. PCA for PDB-reported S:D614G single mutant. PCA results for the whole spike excluding (a-c) and including the 2-up conformations (d-f) in the covariance calculation. (a) Conformational evolution showing RBD opening motions observed in PC1 of the whole spike excluding 2-up conformations is shown from a top view ranging from a closed state (green) to an open state (red). (b) Conformational evolution along PC2 of the whole spike excluding 2-up conformations is shown from a top view from one extreme in green (compressed state) to the other in red (stretched state). Different domains move differently besides the compression and stretching of the structure. (c) Projection of the PDB S:D614G structures (green) and our cryo-EM structures (red and blue) onto the space of the first two PCs of the whole spike excluding 2-up conformations. Note a flipping of PC1 in this analysis relative to the PCAs from MD ensembles. (d) Concerted RBD opening motions observed in PC1 of the whole spike including 2-up conformations are shown from a top view ranging from one extreme in green (closed state) to the other in red (open state). (e) Anticorrelated RBD opening motions observed in PC2 of the whole…