Analyses of the Spike Proteins of Severe Acute Respiratory Syndrome-Related Coronaviruses

Aim: To analyze spike proteins of Severe Acute Respiratory Syndrome (SARS)-related coronaviruses (CoVs) for their conserved motifs, Receptor-Binding Domain (RBD), Receptor Binding Motif (RBM) of SARS-CoV (CoV-1), SARS-CoV-2, Middle East Respiratory Syndrome (MERS)-CoV and their relationship to the bat, pangolin and palm civet-CoVs as possible intermediate hosts.

Study Design: Multiple sequence analysis (MSA) of spike proteins of different SARS-CoVs were studied using Clustal Omega and ExPASy tools.

Methodology: Bioinformatics, SDM and X-ray crystallographic data of the spike proteins from different CoVs including the current epidemic causing SARS-CoV-2 were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different spike proteins from various CoVs and ExPASy tool was used for pI analysis.

Results: Spike proteins in coronaviruses play important roles in mediating receptor binding, membrane fusion, and viral entry into human cells. Furthermore, nowadays all the vaccine development programmes are mainly focused on the SARS-CoV-2 spike protein only, as it plays the crucial, first step in the infection process. Therefore, the spike proteins of the SARS-related coronaviruses, the main determinant of coronavirus host specificity, are analyzed for their conserved motifs, RBD, RBM, etc. The recent epidemic causing strain, SARS-CoV-2, showed 2 dipeptide deletions and 4 peptide insertions ranging from tetra- to hepta-peptides in its spike protein as compared to its predecessor CoV-1. Most of the insertions are also found in the bat and pangolin CoVs except one unique tetrapeptide. The RBM region shows that the bats, pangolins and CoV-2 exhibit very similar to identical sequences. The overall analyses show that the latest SARS-CoV-2 is related to bats and more to pangolin-CoVs suggesting that the pangolins could be possibly the intermediate host. On the other hand, it is found that palm civet RBM sequences are highly related to CoV-1 and not CoV-2. Possibly the novel CoV-2 would have taken three insertions from bats and/or pangolins and the fourth insertion –PRRA- which is unique to SARS-CoV-2 is critically placed just in the S1/S2 cleavage region. The recently discovered G614 mutation (D614→G) in CoV-2, the most prevalent form in the global pandemic now, is found near the RBD towards the C-terminal. Placement of the unique tetrapeptide in the S1/S2 loop region and replacement with more positive charges on the spike protein which resulted in marked increase in the basicity of the SARS-CoV-2 spike protein may, possibly result in significant effects on the structure and function of the protein, possibly leading to rapid transmission.

Conclusions: RBD and RBM regions of the spike proteins of SARS-CoV-1 and palm civet show very close identity to each other whereas the SARS-CoV-2, pangolin- and bat-CoVs exhibit very close identities in their RBD and RBM regions. The two crucial modifications in the spike protein of SARS-CoV-2, viz. a marked increase in the basicity of the protein and the insertion of a dibasic tetrapeptide (–PRRA-) at the critical S1/S2 cleavage point possibly make it to bind to the ACE2 receptor with higher affinity and get it cleaved by the host proteases more efficiently with subsequent effective internalization of the viral genome.