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The rapid rise to dominance of strains carrying the D614G spike mutation of the SARS-CoV-2 virus wherever it has arisen around the world has stimulated much research into the possible survival advantage associated with this genetic variant.
A new study, published on the prepress server bioRxiv* in November 2020 by researchers from the Indian Institute of Science, has explored this issue further. Researchers have found that this mutation is associated with structural and energetic changes that make this spike variant more available during infection, making the associated strain more infectious.
Three variants
The three main circulating variants of SARS-CoV-2 show 1) D614G mutation in the spike protein, 2) G251V in the non-structural protein (NS3), and 3) L84S in the ORF8 protein. The current study focuses on the replacement of D614G.
The SARS-CoV-2 spike glycoprotein comprises three parts: a large part that protrudes from the surface of the virus, an anchor segment, and a small intracellular tail. The protruding part (also known as the ectodomain) has two parts, S1 and S2, that join and fuse with the host cell, respectively.
The two subunits
The S1 / S2 junction has a cleavage site that allows the virus to remove the S1 subunit, while the S2 has another cleavage site that cleaves the S2 region. S1, in the functional protein, is the site of the receptor-binding domain (RBD), which can adopt an “open” or “closed” conformation, depending on its orientation.
In the “open” state, RBD exposes the binding site that binds to the host cell receptor, angiotensin converting enzyme (ACE2), in a peptidase domain. This attachment step keeps the virus in the host cell. This is followed by activation of the S1 / S2 cleavage site by host protease, causing the S1 subunit to shed.
Again, the S2 subunit undergoes a cleavage, which triggers a significant change in the conformation of S2. This causes the virus fusion peptide to insert into the host cell’s cell membrane, forming a spike-shaped protein bridge between the two. The presence of a hairpin loop in S2 brings the two closer together, such that the viral genome is internalized and enters the host cell to produce a viral infection.
The polybasic cleavage site at S1 / S2 ensures that the host furin protease can preactivate the SARS-CoV-2 spike protein while packaging, after its synthesis within the infected host cell. This means that the virus no longer requires the host protease to fuse, speeding up the infection of more cells after the release of new virions from the infected target cell.
Frustration in the local interaction energies of the SD614 and SG614 variants. Mutational (upper panel) and configurational (lower panel) frustrations that exist in interactions between residues formed by aspartate or glycine at position 614 are shown. Minimal and highly frustrated interactions are indicated by green and red lines, respectively. The water-mediated interactions are represented as dashed lines and the variant residue is shown as a sphere. The results for the closed and partially open conformation are shown for one protomer (chain ID: A) in the trimer and similar patterns are observed for the other two protomers (Supplemental Table S1).
Effect of the Spike D614 mutation
Any mutation in the spike protein that alters this first step in viral infection can, therefore, cause a change in the transmission dynamics of the virus or its pathogenicity. In the original strain, position 614 carries the amino acid aspartate, which in D614G is replaced by glycine. This is known to confer greater infectivity on the virus.
At the same time, the presence of glycine instead of aspartate interferes with the formation of a salt bridge between this aspartate and lysine at position 854 of the next protomer. Therefore, this alteration may allow the open state to be more frequent in the mutant.
A cryoelectron microscopy (cryo-MS) study confirms that the presence of glycine at this position stabilizes the peak protein and prevents the S1 subunit from being removed too early.
Frustration and mutation index D614
In the current study, the researchers looked at a measure called the frustration index (FI). They found that in the ancestral variant D614, frustration is high in both the “closed” and “partially open” states.
At G614, however, frustration is neutral in both conformations. This finding shows that the D614G mutation produces a more stable bond between the S1 and S2 subunits of each protomer, as well as between the subunits of adjacent protomers, thus increasing the stability profile of the trimeric peak protein.
Again, the aspartate in D614 comes into contact with other residues within the same protomer and between protomers, using electrostatic or water-mediated bonding. In a total of 8 links, all were very frustrated. With glycine replacing aspartate (G614), in both states, glycine has three minimally frustrated contacts within and between the protomers, with an additional contact in the “partially closed” conformation. The fact that glycine creates minimally frustrated contacts shows an improvement in the environment around the 614 position compared to aspartate.
Aside from this, they looked at how frustrated the original contact between two amino acids was compared to other possible contacts between the two residues involved: configurational frustration. This analysis showed that in the “closed” state, aspartate has only one favorable contact within the same protomer versus the six contacts created within the same protomer and adjacent protomers, with glycine at this position.
When in the ‘partially open’ conformation, there is only one highly frustrated contact with aspartate at 646, but glycine makes four minimally frustrated contacts.
Naturally, reducing the frustration in the local energies of interaction will have a marked effect on the thermodynamic stability of the trimeric peak. The total free energy of the trimer shows a significant reduction in both conformations. This thus improves the stability of the trimeric peak.
Reduced free energy improves infectivity
The researchers summarize, “Overall, single-residue, mutational, and configurational frustrations calculations reveal that glycine substitution changed the local interaction energy in the favorable direction.. “
The result is that the functional peak becomes more available, so it has a higher infectivity, as established by recent studies.
The threat of COVID-19 resurgence globally has created an urgent need to understand and develop strategies to counter the virus as soon as possible. The fact that the D614G strain is dominant wherever it has entered the population means that it has received a lot of attention. The current study focuses on energy reduction in terms of configurational, single residue and mutational frustration associated with this substitution. In doing so, the researchers have found that the significant reduction in free energy and improved stability with a higher frequency of the “partially open” conformation have caused the strain to become highly infectious; more, in fact, than the previous strain.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.
