Mutational Hotspot in the SARS-CoV-2 Spike Protein N-Terminal Domain Conferring Immune Escape Potential

Abstract

Global efforts are being made to monitor the evolution of SARS-CoV-2, aiming for early identification of genotypes providing increased infectivity or virulence. However, viral lineage-focused tracking might fail in early detection of advantageous mutations emerging independently across phylogenies. Here, the emergence patterns of Spike mutations were investigated in sequences deposited in local and global databases to identify mutational hotspots across phylogenies and we evaluated their impact on SARS-CoV-2 evolution. We found a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. These mutations might have an impact on the evasion of neutralizing antibodies. Finally, we found that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path in which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape. We conclude that adaptive mutations, frequently present outside of the receptor-binding domain, can emerge in virtually any SARS-CoV-2 lineage and at any geographical location. Therefore, surveillance should not be restricted to monitoring defined lineages alone.

Keywords: SARS-CoV-2 genome; W152; coronavirus; immune escape; neutralizing antibody; spike NTD; viral evolution.

Figure 1

Diverse W152 mutations emerged independently across numerous genomic contexts. (A) A cumulative number of sequences bearing three W152 mutations uploaded to GISAID (y-axis) depending on the upload date (x-axis). (B) Number of sequences bearing three W152 mutations uploaded weekly to GISAID (y-axis) depending on the upload date (x-axis). (C) Representative phylogenetic tree of analyzed sequences (displaying 1% of the global Audacity tree); 171 independent W152C/L/R clades are identified in the present study and displayed to outline mutation recruitment events (dots) and ensuing contagion clusters (edges); largest cluster for W152C (CAL.20C) is indicated. (D) Fish plot of all SARS-CoV-2 genomes deposited in GISAID, with color-coded areas corresponding to the numeric abundance of clades that recruited W152C (purple—20 events), W152L (turquoise—75 events) or W152R (yellow—76 events) mutations, as a function of the upload date. (E) Plot showing the weekly count of recruitments of clades bearing mutations at positions W152 (black), L452 (orange), E484 (red), and N501 (green); grey areas indicate values for percentiles 75th, 90th, 95th, and 99th of mutation recruitments across all Spike positions.

Figure 2

Most W152 mutation recruitments are not associated with a known adaptive RBD mutation. (A) Plots showing, for the three W152 substitutions, the frequency of co-occurrence of non-synonymous Spike mutations per individual sequence, present at a frequency of at least 0.01; known, adaptive RBD mutations are indicated by orange, red, and green labels. (B) As (A), but calculated per clade, regardless of clade size, shown for frequencies of at least 0.05. (C) Plot showing, for three W152 substitutions, independently recruited clades (circles) colored depending on the co-existing adaptive RBD mutation and positioned depending on the clade size (x axis).

Figure 3

Interactions with neutralizing antibodies are weakened by W152 mutations. (A) Normalized ddG calculation with the wild type Spike and the three W152 mutants (W152C, W152L, W152R) for binding to different anti-NTD nAb, based on available structures. For complexes with 1–87 and 4A8, W152 is found within the binding interface, for 5–24, FC05 and S2X333 complexes W152 is a secondary interaction point at the edge of the binding interface. (B) a zoomed-in view of the W152-containing region for available structures of Spike in complex with anti-NTD nAbs; in all cases, the nAb presents a residue engaged in pi stacking with W152 (red box); aromatic amino acids are the binding partners for W152 in all nAbs except S2X333, where arginine is involved in the interaction. (C,D) Interaction interfaces of Spike W152 mutants with 1–87 and 4A8, respectively.

Figure 4

Extensive NTD evolution during the second wave of the pandemic. (A) Structural model of Spike trimer (PDB Id: 7DDD); RBD marked in green, NTD marked in red; chosen mutation positions are indicated. (B) Spike structure model colored according to the number of mutation recruitments for each amino acid position for weeks 1–55 of the pandemic. The color ramp partitions the number of recruitments into eight equally-spaced bins ranging between the minimum value (blue) and two times the average (light blue); all remaining observations that include more variable amino acid positions are color-coded in yellow. (C) As B, but for weeks 56–75. (D) Boxplot showing mutation recruitment events for all Spike positions present in NTD (red), RBD (green), or other parts of the protein (grey) for weeks 1–55 (left) or 56–75 (right); the statistical significance is assessed using permutation testing (10,000 permutations).