Fig. 1: CTSL cleaves the SARS-CoV-2 S protein at two novel sites.
a Schematic illustration of the SARS-CoV-2 S glycoprotein in which the functional domains and cleavage sites are highlighted (NTD N-terminal domain, RBD receptor-binding domain, CTD C-terminal domain, FPPR fusion-peptide proximal region, HR1 heptad repeat 1, HR2 heptad repeat 2, TMD transmembrane domain). CTSL cleaves at CTSL cleavage site 1 (CS-1) and CTSL cleavage site 2 (CS-2). Furin cleaves at the S1/S2 site. b Overall structure of the SARS-CoV-2 S ectodomain (PDB entry: 6VXX). The CTSL cleavage sites, CS-1 and CS-2, are colored in red, while the furin cleavage site at S1/S2 is colored orange. The three protomers of the S trimer are colored in pink, purple and green. c Schematic illustration and SDS-PAGE analysis for the cleavage of SARS-CoV-2 S glycoprotein. The purified SARS-CoV-2 S protein ectodomain was incubated with different concentrations of CTSL (2–8 μg/mL). The N-terminal sequencing results for band 1 (60 kDa), band 2 (100 kDa), and band 3 (170 kDa) are also shown. All samples were subjected to SDS-PAGE, and bands were detected by Coomassie blue staining. d 4-Gly-mutant SARS-CoV-2 S cannot be cleaved by CTSL into the 60 kDa (band 1) and 100 kDa (band 2) fragments. WT S2p protein (1 μg) and mutant S protein (1 μg) were incubated with CTSL (8 μg/mL). All samples were subjected to SDS-PAGE, and bands were detected by silver staining. e The P1 and P2 residues in CS-1 and CS-2 were mutated to glycine, and the mutant SARS-CoV-2-2-S glycoprotein was named 4-Gly-mutant S. f Amino acid sequence alignment of residues around CS-1 and CS-2 in SARS-CoV-2 variants. P1 and P2 residues that are the same as the WT residues are highlighted in red. The symbol * indicates amino acid residues that are conserved among all tested sequences. VOC variant of concern, VOI variant of interest, VUM variant under monitoring.
