Novel cleavage sites identified in SARS-CoV-2 spike protein reveal mechanism for cathepsin L-facilitated viral infection and treatment strategies

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) "up" activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

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 S_2p 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.

Fig. 2. Cryo-EM structures of CTSL-untreated and -treated SARS-CoV-2 S proteins.

a Side and top views of cryo-EM map of untreated S protein. The three protomers of S protein are shown in light green, yellow, and sky blue. The three RBD domains are highlighted in Sea Green, gold, and deep sky blue, respectively. Glycosylation modifications are colored in tomato. b Side and top views of cryo-EM map of CTSL-treated S protein in different states. The map colors are as same as a. c The four structures of S proteins are superposed together in different colors. CS-1 and CS-2 sites are colored in red and as indicated. The CS-1 and CS-2 regions are zoomed in to show the details. The cleavage sites are indicated by the red lighting-shaped symbol. For untreated S protein, the electron density around the cleavage site is represented as black grid. For the CTSL-treated S, the cleavage site cannot be traced and is represented as dotted line.

Fig. 3. CTSL cleavage sites are essential for SARS-CoV-2 infection and efficient cell–cell fusion.

a Overview of SARS-CoV-2 S proteins with mutations in CS-1, CS-2, and the S1/S2 cleavage site. b Infectivity of PsVs with different point mutations in CS-1 and CS-2 was assessed in LLC-MK2, Vero, Huh7, 293 T/ACE2 cells and in 293 T/ACE2 cells with CTSL (293 T/CTSL), TMPRSS2 (293 T/TMPRSS2), and FURIN (293 T/FURIN) genes overexpression. PsV infectivity was measured by a luciferase assay and is shown as the raw luciferase activity (n = 3–4). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. c–e Quantitative analysis of syncytium formation induced by CS mutant SARS-CoV-2 S proteins (see also Supplementary Fig. S9). c Luciferase gene expression was driven by the ERE promoter, and ESR1 (activator) bound and activated the ERE promoter to upregulate luciferase expression. d, e Effector Huh7 cells were cotransfected with plasmids expressing ERE-luciferase and different S proteins as indicated, and another plate of target Huh7 cells was transfected with plasmid expressing ESR1. After 24 h, the effector cells were detached and added to the target cells for 30–60 min. Then, the supernatant was removed and treated with PBS or CTSL (8 μg/mL) for 20 min. The reaction was stopped by adding 500 μL of medium, and culture was continued for another 24 h to allow cell–cell fusion. When a target cell and effector cell fused to form a syncytium, ESR1 bound and activated the ERE promoter to upregulate luciferase expression. Luciferase activity was then measured as a proxy for the fusion rate. The data were normalized to the WT-PBS group (n = 3). Statistical significance was assessed between the indicated group and the WT-PBS group by two-way ANOVA with Dunnett’s post-hoc test. f Images of syncytium formation induced by CS mutant SARS-CoV-2 S proteins. Huh7 cells were transfected with plasmids to express the WT, CS-1M, CS-2M, or CS-1M + 2 M S protein. Cells were treated in the absence (PBS, pH = 5.8) or presence of CTSL (4 μg/mL, pH = 5.8). Images were acquired after an additional 10–16 h of incubation in medium (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from three independent experiments are shown. g Overexpression or knockdown of the CTSL gene dose-dependently promoted or inhibited, respectively, infection with WT (Wuhan-1) and three mutant SARS-CoV-2 PsVs with different point mutations in the furin cleavage site (FM-delta, FM-ARAA, and FM-GSAS). PsV infectivity in Huh7 cells was measured by a luciferase assay and is shown as the relative luciferase activity (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. h CTSL promoted syncytium formation induced by the FM-ARAA mutant SARS-CoV-2 S protein. Effector cells were cotransfected with ERE-luciferase plasmids and either FM-ARAA S or scramble vectors (Control). Target cells were transfected with ESR1 expression plasmid. After the effector cells and target cells were mixed, the supernatant was removed and treated with PBS or CTSL (8 μg/mL and 16 μg/mL). Luciferase activity was then measured and normalized to that in the control group (n = 4). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. i Huh7 cells were transfected with scramble vector (Control) or FM-ARAA S protein expression plasmid. Cells were treated in the absence or presence of CTSL (2 or 4 μg/mL). Images were acquired after an additional 10–16 h of incubation in medium (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from four independent experiments are shown. The data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.

Fig. 4. CTSL inhibitors prevent infection with SARS-CoV-2 and mutant variant PsVs in vitro.

a Vero E6 cells were pretreated with increasing concentrations of each compound for 16 h and were then infected with different SARS-CoV-2 variant PsVs as indicted. At 24 hpi, infectivity was measured by a luciferase assay. The data were normalized to the average value in vehicle-treated cells and are shown as inhibition rates (n = 3). Statistical significance was assessed between the indicated variant and WT PsV by two-way ANOVA with Dunnett’s post-hoc test. The data are presented as the means ± SEM. *P < 0.05, **P < 0.01. b Vero E6 cells were pretreated with increasing concentrations of each compound for 16 h and were then infected with SARS-CoV-2 at an MOI of 0.01. At 24 h post-infection, viral RNA copies in supernatants were quantified by RT-qPCR. The data were normalized to the average value in vehicle-treated cells and are shown as relative infection percentages. The EC₅₀ values for each compound are indicated. Cell viability was evaluated with a CCK kit (TransGen Biotech) (n = 3). c Ex vivo lung tissues from hACE2 mice or a human donor were infected with SARS-CoV-2 with an inoculum of 1 × 106 PFU/mL for 2 h. Then the inoculum was removed and changed with medium with indicated compounds (10 μM for molnupiravir, 4 μM for E64d, 5 μM for Z-FY-CHO, and 0.4 μM for K777) for another 48 h. Tissues were harvested (without adding compounds) at 2 hpi or 48 hpi to determine the viral growth ability. Tissues were harvested at 48 hpi for quantification of viral RNA (n = 3). Statistical significance was assessed between 2 hpi and 48 hpi by unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was assessed between the indicated drug and 48 hpi by one-way ANOVA with Tukey’s post-hoc test. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001. The data are presented as the means ± SEM.

Fig. 5. CTSL inhibitors prevent SARS-CoV-2 infection in vivo.

a E64d and Z-FY-CHO were administered intraperitoneally at 2 days before infection to 3 dpi as prophylactic treatment, and mice were challenged with 106 PFU at 0 dpi; the two drugs were administered therapeutically at 0–3 dpi. Tissue samples were collected at 4 dpi. b Viral RNA copies in mouse lung and nasal turbinate tissues (n = 5 mice/group). The dotted line indicates the lower limit of detection (LOD). Statistical significance was assessed between the indicated group and control group by one-way ANOVA with Tukey’s post-hoc test. c Representative images from histological analysis of lungs from SARS-CoV-2-infected hACE2 mice at 4 dpi. Magnified views of the boxed regions in each image are shown below the corresponding image. The black arrows indicate inflammatory cell infiltration, the black arrowheads indicate bronchiolar epithelial cell degeneration, and the red arrows indicate alveolar septal thickening. The scale bars are indicated in the figures. d Semiquantitative histological scoring of each lung tissue was performed by grading the severity of bronchiolar epithelial cell damage (0–10), alveolar damage (0–10) and inflammatory cell infiltration in blood vessels and bronchioles (0–10) and summing these scores to calculate the total score. Normal = 0, indeterminate = 1–2, mild = 3–4, moderate = 5–7, severe = 8–10. (n = 3) Statistical significance was assessed between the indicated group and control group by one-way ANOVA with Tukey’s post-hoc test.

Fig. 6. Proposed mechanism by which CTSL promotes SARS-CoV-2 infection.

SARS-CoV-2 S contains S1 and S2 subunits. CS-1 is located in the NTD of the S1 subunit, and CS-2 is located near the S1/S2 site. CTSL cleaves the SARS-CoV-2 S protein: (1) By binding with the S protein on the surface of SARS-CoV-2, CTSL cleaves S at CS-1 and CS-2 sites. (2) The cleavage increases the dynamics of the RBD and makes it accessible to ACE2 for binding. (3) CTSL cleaves CS-2 site to separate S1 and S2 subunits to expose the S2 subunit for membrane fusion. (4) The virus fuses with the membrane of target cell, and the viral genetic material is released into the host cell.