Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh coronavirus known to infect humans. SARS-CoV-2 emerged in the last month of 2019 in Wuhan, China, sparking off the ongoing COVID-19 pandemic. This virus enters the host cells through receptor binding and membrane fusion between the viral and host cell membrane.
A recent study published on the preprint bioRxiv* in June 2020 shows that the predominant virus isolate in any region has a specific mutation that gives it the advantage in viral spread. This is more stable than other forms of the virus, though it does not increase the efficiency of viral binding.
The Spike Protein/ACE2 Interaction
The spike protein or S-protein is responsible for viral receptor binding and membrane fusion, extending through the viral membrane. The S-protein is arranged as trimers, with two subunits, the S1 and S2, each responsible for one aspect of viral infection of the human host cell.
The receptor for the SARS-CoV-2 is the angiotensin-converting enzyme (ACE2) molecule, which is a metalloprotease involved in the renin-angiotensin system. The S-protein has a receptor-binding domain (RBD), which binds the receptor on the host cell. Following this binding, the protein is cleaved at the S1-S2 interface by a furin-like proprotein convertase in the cell that is host to the replicating virus. This is different from the SARS-CoV virus in which this site was cleaved by TMPRSS2 or by lysosomal cathepsins in the target cells.
In either case, the S-protein is then processed further within the S2 subunit to proceed to productive infection.
Predominant Mutation: D614G
Genome analysis of various isolates of the SARS-CoV-2 shows several regions with an increased variation. One such S mutation is the D614G mutation in the C-terminal end of the S1 domain, which is in proximity to the S2 subunit. This variant is found to increase in prevalence with surprising rapidity, becoming the predominant variant in every locality where it is found.
The current study by scientists at the Scripps Research Institute showed that even though this mutation was found only in March, at a low frequency of 26%, being absent among 33 sequences in February, its frequency shot up to 65% and then 70% in April and May, respectively. In other words, this is associated with the faster viral spread, and higher viral loads have also been observed.
On the other hand, the role of this mutation in this change in frequency is not well-defined, since other mutations are also associated, such as those in the nonstructural viral proteins nsp3 and RdRp proteins.
What Does the Mutation Do: The Study
The current study aimed at analyzing the role of the mutation in viral entry so as to determine the effect of this mutation on viral transmission and replication. The S-protein with the original D614 genotype and the G614 genotype (>SD614 and SG614 respectively) were pseudotyped into another virus called the MMLV to produce a pseudovirus. For comparison, the SD614 variant lacking the furin-cleavage motif between the S1/S2 domains was also used.
These viruses were used to transfect cultured cells on which human ACE2 (hACE2) was expressed, or without it.
Increased Infectivity, Reduced S1 Shedding
The researchers found that the cells expressing hACE2 and infected with the G614 virus showed nine times greater efficiency of infection compared to the D614 variant. They then compared the ratio of S1 to S2 domains, to find out if there was any change in the rate of S1 shedding following cleavage at the S1/S2 interface.
They found a much higher ratio of S1 to S2 in the cells infected with the G614 variant. This shows the stabilizing effect of glycine at this locus on the S1/S2 interaction, thus preventing the shedding of S1. Also, the total S1 + S2 in these cells was almost five times higher with the same number of pseudoviral particles, while the S1/S2 ratio was 3.5 times higher, compared to the cell infected with D614.
These results were confirmed in several ways, including the use of virus-like particles. In short, the D614G mutation reduces S1 shedding and increases the amount of S-protein in the virus, thus increasing the viral infectivity.
The next step was to examine if this mutation also conferred more efficient binding with ACE2. They performed an ACE2-hACE2 immunoadhesin binding assay, which binds to the RBD of SARS-CoV-2 with the same affinity as hACE2-Ig. They found that the total S-protein was comparable with the expression of either variant. However, the immunoadhesin bound more to the cells expressing G614 than to those with D614.
SARS-CoV-2 viruses binding to ACE-2 receptors on a human cell, the initial stage of COVID-19 infection, conceptual 3D illustration credit: Kateryna Kon / Shutterstock
This is explained in several ways. Firstly, the G614 might enhance RBD exposure and thus increase the degree of hACE2 binding. Alternatively, it could boost the number of binding sites by reducing S1 shedding. The researchers, therefore, looked for S1 domain shedding using a specific anti-S1 antibody.
They found that the second explanation was more likely because the S1 shedding was reduced with the G614 variant. However, both variants were equally sensitive to neutralizing antibodies.
And finally, the researchers explored the supposition that the mutation would promote the shedding of the S1 subunit, losing a hydrogen bond between the D614 in S1 and T859 in S2. Instead, they found that the latter bonds with Q613 instead, while the presence of glycine at 614 allows the Q613 to be aligned better. Other explanations are also possible, including salt bridge formation between D614 and R646, which could render the S1 conformation unfavorable to S2 binding.
S-Protein Stability Increases Incorporation Into Virions
If the S2 domains exposed by S1 shedding lead to instability in the trans-Golgi network membrane, where the S1/S2 boundary is processed, this could lead to reduced incorporation of the S-protein into the viral particles with the D614 genotype. Another possibility is that the exposed S2 subunits may not allow normal post-translational modification, which means the protein is not fit for incorporation into the virion.
At the same time, increased infectivity does not correspond to increased severity, perhaps because the former is due to the presence of higher levels of functional S-protein with G614, while the latter is affected by other factors. The researchers also suggest that the loss of the S-protein in association with the D614 could be balanced by the increased efficiency of fusion with the unstable S-protein if the host cell is next to another target cell. And finally, it is quite conceivable that small differences in the mechanisms of disease are being overlooked even as small sequence variants are being picked up.
D614G Stabilizes Virus with Furin Cleavage Site
The investigators also point out that the presence of a furin cleavage site at the S1/S2 interface allows the S-protein to be cleaved in the host cells within which it replicates, priming the virions for further infection. This cleavage site is absent in the other SARS viruses, which rely on TMPRSS2 and other endosomal cathepsins within the target cells.
The implication is that this D614G mutation stabilized the SARS-CoV-2 with its unique furin cleavage site, but was not preferred in the earlier viruses because they did not have this site.
The researchers sum up: “We show that an S-protein mutation that results in more transmissible SARS-CoV-2 also limits shedding of the S1 domain and increases S-protein incorporation into the virion.” More study will determine how this change affects the disease process or severity, since this change does not affect ACE2 binding or neutralization efficiency.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.