By Sally Robertson, B.Sc

Researchers at the Indian Institute of Technology, Guwahati, have made an important discovery about a viral fusion protein found on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that could help researchers develop a vaccine.

Their finding that a mutation in the protein’s cleavage site disrupts binding to host cells could be used to develop an attenuated version of the virus.

Coronavirus entry to host cells
Coronavirus enters host cells via receptor binding and proteolytic cleavage of the viral spike protein into subunits, which helps it fuse with the cell membrane and release its genome into the cytoplasm.

The proteases cathepsin B, plasmin, trypsin, elastase, and transmembrane protease/serine have previously been demonstrated to cleave the protein as part of the viral attachment process. This activation of the spike protein enables glycoproteins on the viral envelope to fuse to host cells and enable virus entry.

The ectodomain of the spike protein is made up of subunit S1, which is involved in receptor binding and subunit S2, which maintains fusion.

Researchers already know that in the case of SARS-CoV, sequence motifs lying between S1 and S2 determine the sites that host cell proteases bind to and cleave.

What has the current study involved?
Now, Sachin Kumar and colleagues have demonstrated the binding of host-cell proteases plasmin, furin, and cathepsin B to the SARS-CoV-2 spike protein.

They have also analyzed the role that certain amino acid residues play in the binding interaction by introducing single point mutations at the protein’s cleavage site.

A preprint version of the paper can be accessed at bioRxiv, while the article undergoes peer review.

The study results suggested the inclusion of four additional polybasic amino acids at the S1/S2 boundary, that point to a role of furin as a host proteolytic activator of the spike protein.

Molecular docking analysis (which determines the interaction between two molecules) of host-cell proteases found that they bind to the protein by forming salt bridges and hydrogen bonds. To test the role that these additional amino acid residues play in the interaction, the basic amino acid was sequentially mutated to alanine.

“Further, we analyzed the binding efficiency of each of the mutants with respective host protease,” writes Kumar and colleagues.

Understanding the role of individual residues
First, the team established all residues involved in the binding of plasmin, furin, and cathepsin B to wild-type spike protein.

The docking study revealed important information about the interaction between cathepsin B and the proteolytic cleavage site.

The interaction was disrupted when a single residue was mutated at the protein’s cleavage site. All mutant models had fewer salt bridges and hydrogen bonds, thereby suggesting weaker binding.

In particular, “mutation of P682A in the wild type S [spike] protein results in the best mutant model that blocks the enzyme active site for cellular proteases, owing to a minimum number of salt bridge and hydrogen-bond formation,” writes the team.

Furthermore, “the finding suggested that PRRARS amino acid motif in the type S protein are responsible for its proper binding with furin, cathepsin and plasmin host proteases and mutation of these residues impair its interaction.”

The authors say that cleavage of arginine is commonly used to activate a number of proteins, including hormones and growth factors.

They add that the docking studies suggest that modifications made at these particular sites weaken the binding of host-cell proteases to the spike protein.

Vaccine development studies
“The participation of these crucial amino acids located at the boundary of S1 and S2 subunits in the proteolytic processing step will provide a unique opportunity to develop a lower pathogenic strain of SARS-CoV-2, which can further be used for vaccine development studies,” said Kumar and colleagues.

“Considering the fact proven by this in silico analysis that the single amino acid substitutions can help to make an attenuated form of virus, further in vitro work is needed to validate the fact,” they suggest.

Kumar and the team also point out that the use of protease inhibitors may represent a new approach to stopping the cellular spread of SARS-CoV-2 and to developing an antiviral.

Journal reference:
Kumar S, et al. Insight towards the effect of the multibasic cleavage site of SARS-CoV-2 spike protein on cellular proteases. bioRxiv 2020. doi: https://doi.org/10.1101/2020.04.25.061507