The study team reported 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.
The study team investigated the impact these mutations might have on the evasion of neutralizing antibodies and shockingly discovered that the NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path on which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape.
The study findings were published on a preprint server and are currently being peer reviewed.
Globally, the selective pressure imposes conservation of adaptive mutations facilitating the viral spread. The overall success of viral transmission depends on the mutation rate, the extent of immune response, and the population size (3). During the pandemic, where population size is large, rapid increase in the frequency of alterations is observed at critical positions of the viral genome.
Two commonly reported forces shaping the natural selection for SARS-CoV-2 are the adaptation to host (4) and the evasion of the immune response (5), including immunity triggered by the vaccines (6). Consequently, the evolutionary rate is particularly high for the S gene encoding the Spike protein (7), the main contact point with the ACE2 receptor of the host cell (8). Importantly, Spike serves also as the immunizing agent in the majority of COVID-19 vaccines (9).
It is expected that mutations improving viral fitness emerge independently across unrelated viral clades. An example of an adaptive mutation that emerged relatively early during the pandemic is D614G substitution in Spike, by the end of 2020 present in almost every SARS-CoV-2 genome in the world (10) and believed to improve the Spike trimer interaction with ACE2 (4,11).
Since the last months of 2020, increase in frequency of other mutations was observed, with the N501Y and E484K being two prominent examples. The mechanisms by which they confer evolutionary advantage to SARS-CoV-2 vary. Particularly, N501Y increases the adaptation to host by enhancing interaction with the ACE2 receptor (12–14) resulting in more efficient transmission (15).
In contrast, E484K appears as selectively advantageous by decreasing the strength of interaction with neutralizing antibodies (5,16,17), which facilitates evasion of the immune response. More recently, L452R substitution was reported to have similar properties to E484K (5,16,18,19). Importantly, these mutations have arisen independently within diverse, unrelated genomic contexts, and at distant geographical locations, being examples of convergent evolution.
Moreover, it may be expected that certain genomic positions under strong negative frequency-dependent selection – as expected in the context of immunity-escaping processes (20) – will display a diverse spectrum of mutations.
Adaptive traits require close monitoring, particularly because they are likely to appear as increasingly prominent within SARS-CoV-2 strains under the current global vaccination efforts aiming at establishing herd immunity. Several studies focused on evaluating potential impact of mutations on the viral spread and antibody evasion (16,21–27).
Most investigations focused on the receptor binding domain (RBD) of the Spike, the immunodominant part of the protein (28) containing the ACE2-interacting interface. However, mutations at sites outside of the RBD, such as D614, might also have strong impact on both, the infectivity and immune escape. For example, the N-terminal domain (NTD) of the Spike was shown to be a potent target for neutralizing antibodies (6,29,30).
By screening SARS-CoV-2 genome sequences for residues undergoing frequent and diverse mutations we pinpointed W152, a residue present in NTD, whose alterations have the potential of being advantageous for viral transmission. We identified that several substitutions, leading to a limited set of amino-acid changes at position W152, were independently recruited numerous times across many distantly related phylogenetic contexts and diverse geographical locations, suggesting their adaptive character. Insights from structural studies confirm that the identified W152 substitutions remove an important interaction point for multiple potent neutralizing antibodies.
Furthermore, we demonstrate that mutations in NTD were recruited more frequently than in other regions of Spike during the second wave of the pandemic, likely due to improving viral fitness through the immune escape. Our work highlights the importance of monitoring individual mutations occurring outside of the Spike RBD.
Accumulating body of evidence suggests a key role of Spike NTD mutations in viral evolution. Based on conservation estimates and the number of independent mutation emergences we demonstrate that this domain undergoes more rapid evolutionary changes in comparison with other parts of the protein. This localized evolution gained momentum during the second wave of the pandemic, likely in response to global increase in immunity.
The most prominent forces driving selection of Spike mutations are the increased interaction with ACE2 and the evasion of neutralizing antibodies (nAb). NTD and RBD constitute the most exposed parts of the Spike making them the most likely targets of the immune response. As demonstrated by us and others, mutations in these domains often facilitate immune evasion (5,6,16,17,21,24,25,36).
However, RBD mutations are more evolutionarily constrained due to their role in interaction with ACE2.
Given the relatively small contribution of NTD to ACE2 binding, alterations in this domain might constitute the evolutionary ‘disguise’ the virus uses to avoid antibody neutralization. The variability is not restricted to amino acid substitutions as a significant number of deletions was also reported in NTD and linked to the immune escape (42).
Identification of potential nAb that can be used against different variants of RBD and NTD is of great importance, considering that antibody cocktails might act collaboratively to impede the progression of viral infection (37).
We identified W152 as a NTD residue undergoing particularly extensive evolutionary dynamics, highlighted by multiple individual substitutions emerging across many phylogenetic and geographical contexts.
Remarkably frequent mutation recruitment events were reported at this position globally, with a clear increase in intensity since the end of 2020 (week 55 of the pandemic).
The largest clusters reported for each of the three frequent substitutions – W152L, W152R or W152C – were characterized by the co-recruitment of one of the prominent, adaptive RBD mutations (E484K, N501Y or L452R, respectively). Although the contribution of W152 mutations to those three particular events could not be decoupled from that of their co-occurring alterations in RBD, our results suggest an adaptive role of the W152 substitutions as most of their recruitment events did not occur in parallel with RBD mutations. In line with this finding, recent study demonstrated that W152C allows to further increase B.1.429 infectivity in comparison to L452R alone (18).
It is generally appreciated that advantageous mutations initially arise as the quasi-species, present only in a fraction of viral genomes within a given host. Providing a competitive edge, they are progressively increasing in prevalence and are eventually transmitted to new hosts giving rise to new clades responsible for infection clusters. In this regard, the advantage conferred by W152 mutations might be exemplified by a reported increase in the intra-host fraction of genomes bearing W152L substitution during the infection (43).
Tryptophane at position 152 was shown to have a role in stabilizing intermolecular interactions of the Spike (44). Our analysis suggests that mutations of W152 diminish the strength of interactions with several neutralizing antibodies, consistent with other reports (6,29,36).
As the chemical properties of each side chain of the amino acids present in the mutants differ (leucine – hydrophobic, arginine – basic, cysteine – sulfide) the advantage likely stems from the fact of removing tryptophane (aromatic) from the relevant position rather than establishing specific novel interactions.
Such alteration decreases the propensity of forming stable stacking interactions similar to those observed in the 1-87 or 4A8 antibody complexes with Spike (29). Alarming evidence supporting the role in W152 in evading immune response comes from a report of the R.1 lineage bearing W152L (45) being responsible for a local outbreak in a population having underwent a vaccination program (46).
Most SARS-CoV-2 evolution studies concentrate on the mutations present in RBD. Our work clearly suggests that large emphasis should be also put on monitoring alterations occurring in NTD. Dedicated biochemical, immunological and cell biology investigations are necessary to understand the exact effects of the W152 substitutions on Spike properties as well as on SARS-CoV-2 infectivity or virulence.
Significant efforts are spent on tracking the spread of specific variants of concern, such as the B.1.1.7 (“UK variant”) or the B.1.167.2 (“Indian variant”). However, our study outlines the importance of monitoring the emergence, recruitment and spread of individual mutations as well. Mutations of residues such as W152, L18, H69, Y144, L452, E484 or N501 occurred frequently and have been recruited across many independent SARS-CoV-2 lineages.
As of writing, the W152 mutations spurred 171 independent clusters that directly or indirectly contributed to contaminate over 15,000 patients. Hence, monitoring efforts should not overlook the fact that evolutionary advantageous mutations can emerge in virtually any SARS-CoV-2 lineage and at any geographical location.