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Abstract

Recent research on the SARS-CoV-2 pandemic has exploded around the furin-cleavable polybasic insert PRRAR↓S, found within the spike protein. The insert and the receptor-binding domain, (RBD), are vital clues in the Sherlock Holmes-like investigation into the origin of the virus and in its zoonotic crossover. Based on comparative analysis of the whole genome and the sequence features of the insert and the RBD domain, the bat and the pangolin have been proposed as very likely intermediary hosts. In this study, using the various databases, in-house developed tools, sequence comparisons, structure-guided docking, and molecular dynamics simulations, we cautiously present a fresh, theoretical perspective on the SARS-CoV-2 virus activation and its intermediary host. They are a) the SARS-CoV-2 has not yet acquired a fully optimal furin binding site or this seemingly less optimal sequence, PRRARS, has been selected for survival; b) in structural models of furin complexed with peptides, PRRAR↓S binds less well and with distinct differences as compared to the all basic RRKRR↓S; c) these differences may be exploited for the design of virus-specific inhibitors; d) the novel polybasic insert of SARS-CoV-2 may be promiscuous enough to be cleaved by multiple enzymes of the human airway epithelium and tissues which may explain its unexpected broad tropism; e) the RBD domain of the feline coronavirus spike protein carries residues that are responsible for high-affinity binding of the SARS-CoV-2 to the ACE 2 receptor; f) en route zoonotic transfer, the virus may have passed through the domestic cat whose very human-like ACE2 receptor and furin may have played some role in optimizing the traits required for zoonotic transfer.

Keywords: Feline CoV; Furin; Host; Proteases; RBD; SARS-CoV-2; Spike protein.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. SK and CSV are founder directors of Sinopsee Therapeutics and Aplomex; neither company has any conflict with current work.

Figures

Fig. 1
Fig. 1
Frequency of distribution of the ‘putative P5 residue’ in proteins that may be substrates of furin and furin-like enzymes as they carry the RRARS motif. Proteins carrying the perfect match for this cleavage sequence RRARS were obtained using the program BLAST.
Fig. 2
Fig. 2
Frequency of amino acid residues found at the P2 position of all the known cleavage sequences of furin and furin-like enzymes. The cleavage sequences of the form P4P3P2P1P1′P2′P3′P4′ archived in the MEROPS database were extracted. Each amino acid present at the P2 position was counted and normalized to the total observed sites. Note that the basic residues R/K contribute to about 80% of the P2 residues.
Fig. 3
Fig. 3
Snapshots of conformations sampled during the MD simulations of (A) furin-PRRARS (B) furin–RRKRRS complexes. The furin protein is shown as a cartoon (grey) and the bound peptides are shown as a cartoon (green), with the peptide-protein-interacting residues and hydrogen-bond interactions highlighted in sticks and dashed lines, respectively. The three catalytic residues (His194, Asp153, and Ser368) are colored pink. P1'S is labelled as S6 (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
PCs (PSCK) and trypsin like enzymes that can cleave the S1/S2 boundary and activate the virus. A more flexible tetra peptide query set derived from cleavage sites of Serine proteases deposited in MEROPS was used to scan the SARS-CoV-2 spike protein. The logic was based on our program PNSAS created to identify proteolytic sites on natural substrates. The sequence matched sites, accessible on the surface of the S1 domain are indicated.
Fig. 5
Fig. 5
Frequency plot of P4R, P3R, P2A, P1R, and P1′S within the known cleavage sites of the trypsin-like enzymes. The cleavage sequences of the form P4P3P2P1P1′ archived in the MEROPS database for the trypsin-like enzymes (X-axis) were extracted. Each amino acid present at these positions were counted. The number of times the individual amino acids of the polybasic insert RRARS occur at the respective position were normalized to the total observed sites.
Fig. 6
Fig. 6
Phylogenetic map of coronaviruses based on the sequence homology at the S2 domain of the Spike protein. Curated UniProt sequences of Coronavirus were clubbed with the Feline spike protein sequences reported in Ref. [9] and the phylogenetic tree was constructed according to Multiple Sequence Alignment (please refer to methods for details).
Fig. 7
Fig. 7
Interface of the SARS-CoV-2 RBD and the ACE2 receptor complex compared with the sequence of the feline ACE2 and feline CoV ‘RBD domain’. The crystal structure (PDB ID: 6M0J) was used to obtain the interaction map in the 2-dimensional format using PDBsum. Using the pairwise sequence alignment (in Supplementary File 2 – for ACE2 and Supplementary File 3 – for Spike proteins), we juxtaposed the corresponding residues from feline ACE2 and feline CoV CoV RBD domain. Tyr 505, Lys417 of the RBD domain are absolutely conserved and Asn 501 is replaced by Gln. The strain in which this residue has undergone mutation to Tyr has been identified. The charged residues Asp30, Lys31, Lys353, Asp355 of ACE2 are absolutely conserved.
Fig. 8
Fig. 8
The polybasic insert in different isolates of feline corona viruses. The AJO27013.1, AJO26993.1, AJO27003.1 are from spike proteins of SARS isolated from healthy cats. AJO27023.1, AJO26973.1, and AJO26983.1 are from infected cats and belong to the FIPV isolates associated with systemic infection and lethality. RRARR↓S at the S1/S2 boundary found in isolates from healthy cats is one of the well-optimized furin cleavage sequences. This is mutated to a sequence that cannot be cleaved by Furin or has been lost in the FIPV strains.
All figures (8)
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