Covid-19: why is the D614G mutation more stable and able to spread faster?

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The fast-spreading UK, South Africa, and Brazil coronavirus variants are raising both concerns and questions about whether COVID-19 vaccines will protect against them.

New work led by Bing Chen, Ph.D., at Boston Children’s Hospital analyzed how the structure of the coronavirus spike proteins changes with the D614G mutation – carried by all three variants – and showed why these variants are able to spread more quickly. The team reports its findings in Science (March 16, 2021).

Chen’s team imaged the spikes with cryo-electron microscopy (cryo-EM), which has resolution down to the atomic level. They found that the D614G mutation (substitution of in a single amino acid “letter” in the genetic code for the spike protein) makes the spike more stable as compared with the original SARS-CoV-2 virus. As a result, more functional spikes are available to bind to our cells’ ACE2 receptors, making the virus more infectious.

Preventing spikes’ shape change

In the original coronavirus, the spike proteins would bind to the ACE2 receptor and then dramatically change shape, folding in on themselves. This enabled the virus to fuse its membrane with our own cells’ membranes and get inside. However, as Chen and colleagues reported in July 2020, the spikes would sometimes prematurely change shape and fall apart before the virus could bind to cells. While this slowed the virus down, the shape change also made it harder for our immune system to contain the virus.

A sturdier spike protein explains the faster spread of coronavirus variants
This model shows the structure of the spike protein in its closed configuration, in its original D614 form (left) and its mutant form (G614). In the mutant spike protein, the 630 loop (in red) stabilizes the spike, preventing it from flipping open prematurely and rendering SARS-CoV-2 more infectious. Credit: Bing Chen, PhD, Boston Children’s Hospital

“Because the original spike protein would dissociate, it was not good enough to induce a strong neutralizing antibody response,” says Chen.

When Chen and colleagues imaged the mutant spike protein, they found that the D614G mutation stabilizes the spike by blocking the premature shape change. Interestingly, the mutation also makes the spikes bind more weakly to the ACE receptor, but the fact that the spikes are less apt to fall apart prematurely renders the virus overall more infectious.

“Say the original virus has 100 spikes,” Chen explains. “Because of the shape instability, you may have just 50 percent of them functional. In the G614 variants, you may have 90 percent that are functional, so even though they don’t bind as well, the chances are greater that you will have infection.”

Chen proposes that redesigned vaccines incorporate the code for this mutant spike protein. The more stable spike shape should make any vaccine based on the spike (as are the Moderna, Pfizer, and Johnson & Johnson vaccine) more likely to elicit protective neutralizing antibodies, he says.

Future direction: A drug to block coronavirus entry

Chen and his colleagues are further applying structural biology to better understand how SARS-CoV-2 binds to the ACE2 receptor, with an eye toward therapeutics to block the virus from gaining entry to our cells.

In January, the team showed in Nature Structural & Molecular Biology that a structurally-engineered “decoy” ACE2 protein binds the virus 200 times more strongly than the body’s own ACE2. The decoy potently inhibited the virus in cell culture, suggesting it could be an anti-COVID-19 treatment. Chen is now planning to advance this research into animal models.


A novel variant of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on wild-type human-codon-optimized Spike to introduce the D614G variant.

Using multiple human cell lines, including human lung epithelial cells, we found that the lentiviral particles pseudotyped with Spike D614G are more effective at transducing cells than ones pseudotyped with wild-type Spike. The increased transduction with Spike D614G ranged from 1.3- to 2.4-fold in Caco-2 and Calu-3 cells expressing endogenous ACE2 and from 1.5- to 7.7-fold in A549ACE2 and Huh7.5ACE2 overexpressing ACE2.

Furthermore, trans-complementation of SARS-CoV-2 virus with Spike D614G showed an increased infectivity in human cells. Although there is minimal difference in ACE2 receptor binding between the D614 and G614 Spike variants, the G614 variant is more resistant to proteolytic cleavage, suggesting a possible mechanism for the increased transduction.

Discussion
In summary, we have demonstrated that the recent and now-dominant mutation in the SARS-CoV-2 Spike glycoprotein D614G increases the efficiency of cellular entry for the virus across a broad range of human cell types, including cells from lung, liver, and colon. We showed increased entry efficiency using both a pseudotyped lentiviral model system and a replication-competent SARS-CoV-2 virus. Given the concordance between the pseudotyped lentiviral system and SARS-CoV-2 virus, this suggests that changes in Spike protein are well represented using the pseudovirus, which should enable a much broader group of laboratories to use and study Spike variants.

We also found that G614 Spike is more resistant to proteolytic cleavage during production of the protein in host cells as well as on pseudotyped lentiviral particles, suggesting that replicated virus produced in human cells may be more infectious due to a greater proportion of functional (uncleaved with receptor-binding domain) Spike protein per virion.

In contrast to a recent study with Spike-pseudotyped murine leukemia virus (Zhang et al., 2020), we did not observe a difference in Spike incorporation into lentiviral particles with the G614 variant as compared to the D614 variant, which is consistent with observations by other groups (Ozono et al., 2020; Yurkovetskiy et al., 2020).

It is now well established that SARS-CoV-2 Spike is proteolytically processed primarily by furin; however, new reports suggest that G614 maybe also processed by elastase-2 (Hu et al., 2020). Future research is required to confirm these findings and establish if Spike G614 is proteolytically processed differently on live SARS-CoV-2 virus.

Using bio-layer interferometry with purified SARS-CoV-2 Spike S1 fragment monomers and ACE2 proteins, we found no significant difference in binding kinetics with the ACE2 receptor resulting from the D614G mutation. Yurkovetskiy and colleagues reported that, with purified Spike full-length trimers, the G614 mutation shifts the Spike protein conformation to an ACE2-binding competent state (Yurkovetskiy et al., 2020), suggesting that Spike full-length trimers behave differently than the Spike S1 monomers that we investigated in this study.

Several other groups have also reported that the D614G results in increased viral fitness and infection efficiency (Hu et al., 2020; Jiang et al., 2020; Li et al., 2020; Ozono et al., 2020; Plante et al., 2020; Yurkovetskiy et al., 2020; Zhang et al., 2020). Most of them utilized Spike-pseudotyped viral particles to demonstrate this finding. While the common consensus is that Spike G614 mutation increases viral fitness and infectivity, the mechanism by which it occurs varies among these studies.

Several of these studies investigated the proteolytic processing, incorporation, and ACE2-binding conformation state of the Spike protein, with some discrepancies between them regarding the proteolytic processing and incorporation of Spike. This can be explained, at least in part, by the technical differences in the protocols used to generate the Spike-pseudotyped viral particles.

For example, the studies use a wide variety of pseudoviral systems, such as murine leukemia virus (Zhang et al., 2020), mouse sarcoma virus (Jiang et al., 2020), and lentivirus (our study and Yurkovetskiy et al., 2020). While all four studies utilized HEK293 cells to generate the particles, each one used different transfection methods and it is possible that the transfection reagents can adversely affect the cell state and protease activity.

Two studies that utilized isogenic SARS-CoV-2 D614 and G614 variants found no difference in Spike protein cleavage and incorporation into viral particles (Hou et al., 2020; Plante et al., 2020). The differences in Spike biology between isogenic pseudoviruses and SARS-CoV-2 viruses may be due to differences in Spike protein trimer assembly and presentation, as well as the absence/presence of additional SARS-CoV-2 proteins. Altogether, these differences illustrate the advantages and limitations of using pseudoviruses and isogenic viruses that should be taken into consideration in future studies.

Despite the emerging consensus the G614 results in faster viral spread (Korber et al., 2020), it is still uncertain whether this will have a clinical impact on COVID-19 disease progression. Two studies that have examined potential differences in clinical severity or hospitalization rates did not see a correlation with Spike mutation status (Korber et al., 2020; Wagner et al., 2020), although one study found a small but not significant enrichment of G614 mutations among intensive care unit patients (Korber et al., 2020).

Given its rapid rise in human isolates and enhanced transduction across a broad spectrum of human cell types, the G614 variant and other recent variants merit careful consideration by biomedical researchers working on candidate therapies, such as those to modulate cellular proteases, and on vaccines that deliver Spike D614 nucleic acids or peptides.

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891930/


More information: Jun Zhang et al. Structural impact on SARS-CoV-2 spike protein by D614G substitution, Science (2021). DOI: 10.1126/science.abf2303

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