Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

Abstract

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Fig. 1

Circulating CTSL is elevated in patients with COVID-19. A total of 87 patients with COVID-19, including 20 with severe disease and 67 with nonsevere disease, and 125 healthy volunteers were enrolled in this study. a Plasma CTSL, CTSB, and CTSL/CTSB levels patients with severe (n = 20) and nonsevere COVID-19 (n = 67) upon hospital admission (day 0). Statistical significance was assessed by the Mann–Whitney U test (two-sided). b Plasma CTSL, CTSB and CTSL/CTSB levels in COVID-19 patients (day 0) (n = 87) and age-/sex-matched healthy volunteers (n = 125). The green lines in panels a and b indicate the reference ranges for each parameter, established as the mean values ± 2 SD in the healthy participants. Statistical significance was assessed by the Mann–Whitney U test (two-sided). c Correlation between CTSL in plasma from COVID-19 patients (n = 87) and the number of days from symptom onset to blood collection before therapy. Statistical significance was assessed by Spearman correlation analysis (two-sided). d Flowchart of the follow-up study. Patients were admitted to the hospital on day 0 and experienced a mean hospitalization time of 14 days (day 14). Then, they were followed up on the 14th day (day 28) and the 28th day (day 42) after discharge from the hospital. Blood samples were collected on days 0, 28, and 42. e CTSL levels in plasma from COVID-19 patients (n = 87) on days 0, 28, and 42 after enrollment. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. f Comparison of plasma CTSL levels between patients with nonsevere (n = 67) and severe COVID-19 (n = 20) on days 0, 28, and 42. The data are shown as the medians ± interquartile ranges. Statistical significance was assessed by the Mann–Whitney U test (two-sided). g Summary forest plot of candidate predictor variables associated with the severity of COVID-19 (n = 87) by univariable logistic regression. h Summary forest plot of candidate predictor variables associated with COVID-19 severity (n = 87) in multivariable logistic regression. The predictor variables used in the final model were hypertension, diabetes, sex, age, Ang(1–7), ACE2, CTSB and CTSL

Fig. 2

CTSL is elevated in SARS-CoV-2 pseudovirus-infected cells in vitro. a Schematic of the validation assay setup. Huh7 cells were infected with different doses of SARS-CoV-2 pseudovirus (from 0.047 × 10⁴ TCID₅₀/ml to 1.30 × 10⁴ TCID₅₀/ml). Cells not infected with pseudovirus were used as controls. Luciferase activity and VSV-P mRNA levels were measured to evaluate infection severity. The mRNA and protein levels of CTSL and CTSB in Huh7 cells were measured to validate the clinical data. b, c Luciferase activity (n = 4) (b) and VSV-P mRNA (n = 8) (c) levels increased dose-dependently 24 h after pseudovirus infection. Statistical significance was assessed by Brown–Forsythe and Welch’s ANOVA with Dunnett’s post hoc test in b by with the Kruskal–Wallis test with Dunn’s post hoc test for c. d, e Effects of SARS-CoV-2 pseudovirus infection on CTSL and CTSB mRNA levels (d) and protein levels (e). n = 6. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. Data are expressed as the mean ± s.e.m. values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

CTSL knockdown or overexpression affects pseudovirus infection in vitro. a Schematic of the CTSL knockdown and overexpression assay setup. b Dose-dependent knockdown of CTSL by siRNAs without affecting CTSB expression. n = 4. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. c–e Knockdown of CTSL dose-dependently inhibited SARS-2-S-driven entry, as measured by a luciferase assay and shown as absolute luciferase activity (n = 8) (c) and relative luciferase activity (n = 8) (d) values, and VSV-P mRNA levels (n = 6) (e). Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. f Dose-dependent overexpression of CTSL with a plasmid encoding the CTSL gene without affecting CTSB expression (n = 5). Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. g–i Overexpression of CTSL dose-dependently promoted SARS-2-S-driven entry, as measured by a luciferase assay and shown as absolute luciferase activity (g) and relative luciferase activity h, values, and VSV-P mRNA levels (i) n = 5. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. The data are expressed as the mean ± s.e.m. values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

CTSL cleaves the SARS-CoV-2 spike (S) protein, and this cleavage promotes cell–cell fusion. a Overview of the SARS-CoV-1 and SARS-CoV-2 S1/S2 cleavage sites. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are units of the S2 subunit that function in membrane fusion. b Analysis of CTSL-mediated S-protein cleavage. Purified SARS-CoV-1 or SARS-CoV-2 S protein was incubated in the presence or absence (assay buffer, pH = 5.5) of CTSL (2 or 10 μg/ml in assay buffer, pH = 5 .5) at 37 °C for 1 h. The reaction system of 2 μg/ml CTSL was further supplemented with CTSL inhibitors (20 μM E64d or 20 μM SID 26681509), as indicated. Proteins were subjected to SDS-PAGE and detected by silver staining. Representative data from three independent experiments are shown. c Syncytium-formation assay: Huh7 cells were untransfected (Null) or transfected with plasmid to express the SARS-CoV-2 S protein. Cells were incubated in the presence or absence (PBS, pH = 7.4) of trypsin (2 μg/ml in PBS, pH = 7.4) or in the presence or absence (PBS, pH = 5.8) of CTSL (2 or 4 μg/ml in PBS, pH = 5.8) for 20 min. Images were acquired after an additional 16 h incubation in the medium. (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from seven independent experiments are shown. d Quantitative analysis of syncytia in panel c. n = 7. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are expressed as the mean ± s.e.m. values. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

CTSL inhibitors block SARS-2-S-driven cell entry in vitro. a Schematic of the CTSL inhibitor assay setup. Huh7 cells were pretreated with different drugs 1 h before infection with different pseudoviruses, as indicated, at the same dose (1.3 × 10⁴ TCID₅₀/ml). Pseudovirus infection and cell viability were evaluated by luciferase activity and MTT assay, respectively. b–d Inhibition of SARS-2-S-driven cell entry by different doses of SID 26681509 (b), E64d (c), and amantadine (d) and viability of cells treated with different doses of the drugs as indicated. n = 4. e–g Effects of E64d on SARS-CoV-1 pseudovirus (e), VSV pseudovirus (f), and RVF pseudovirus (g) infection and viability of cells treated with different doses of E64d as indicated. n = 4. h–j Effects of amantadine on SARS-CoV-1 pseudovirus (h), VSV pseudovirus (i), and RVF pseudovirus (j) infection and viability of cells treated with different doses of amantadine as indicated. n = 4. k, l Effects of drug pretreatment on CTSL enzyme activity in Huh7 cells with or without pseudovirus infection. Huh7 cells were pretreated with vehicle, 30 μM E64d (k) or 300 μM amantadine (l) for 1 h and were then infected with SARS-CoV-2 pseudovirus at a dose of 1.3 × 10⁴ TCID₅₀/ml. Cells not infected with pseudovirus were used as controls. n = 7. Statistical significance was assessed by two-way ANOVA with the Holm–Sidak post hoc test for multiple comparisons. The data are expressed as the mean ± s.e.m. values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

CTSL inhibitors prevent pseudovirus infection in humanized mice. Human ACE2 transgenic mice were randomly divided into four groups and pretreated with vehicle or different drugs (E64d or amantadine) as indicated 2 days prior to virus inoculation via tail vein injection (1.5 × 10⁶ TCID₅₀ per mouse). Mice without pseudovirus inoculation were used as the healthy control group. Bioluminescence was measured 1 day post infection and visualized in pseudocolor. a The relative intensities of emitted light are presented as the photon flux values in photon/(sec/cm²/sr) and displayed as pseudocolor images, with colors ranging from blue (lowest intensity) to red (highest intensity). b Pseudovirus infection in each group as indicated by the total flux values. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. c Pseudovirus infection as indicated by the liver VSV-P mRNA levels in each group. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons. d Hepatic CTSL protein levels in each group. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. e Hepatic CTSB protein levels in each group. Statistical significance was assessed by the Kruskal–Wallis test with Dunn’s post hoc test. f Proposed mechanism of CTSL action in SARS-CoV-2 infection. (1) CTSL cleaves the SARS-2-S protein and releases the virus from the endosome. (2) SARS-CoV-2 promotes CTSL gene transcription and enzyme activity through unknown mechanisms. (3) Upregulation of CTSL, in turn, enhances SARS-CoV-2 infection. n = 5. The data are expressed as the mean ± s.e.m. values. *P < 0.05, **P < 0.01