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. 2005 Aug 16;102(33):11876-81.
doi: 10.1073/pnas.0505577102. Epub 2005 Aug 4.

Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

Graham Simmons  1 , Dhaval N Gosalia, Andrew J Rennekamp, Jacqueline D Reeves, Scott L Diamond, Paul Bates
Affiliations

Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

Graham Simmons et al. Proc Natl Acad Sci U S A. .

Abstract

Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currently no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) glycoprotein, and infection is sensitive to lysosomotropic agents that perturb endosomal pH. We demonstrate here that the lysosomotropic-agent-mediated block to SARS-CoV infection is overcome by protease treatment of _target-cell-associated virus. In addition, SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. A cell-free membrane-fusion system demonstrates that engagement of receptor followed by proteolysis is required for SARS-CoV membrane fusion and indicates that cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These results suggest that SARS-CoV infection results from a unique, three-step process: receptor binding and induced conformational changes in S glycoprotein followed by cathepsin L proteolysis within endosomes. The requirement for cathepsin L proteolysis identifies a previously uncharacterized class of inhibitor for SARS-CoV infection.

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Figures

Fig. 1.
Fig. 1.
Effect of trypsin on SARS-CoV infection. (A) Trypsin treatment bypasses ammonium chloride inhibition. HIV-luc(SARS S) or HIV-luc(VSV-G) were bound to mock (black and gray bars) or ammonium chloride-treated (third set of bars and white bars) 293T/ACE2 cells. The cells were incubated with either PBS (black bars and third set of bars) or TPCK-trypsin (gray and white bars). The results are presented as a percentage of no-ammonium-chloride (NH4Cl), no-trypsin (Tryp.) controls (≈4,000 and 10,000 RLU for SARS S and VSV-G, respectively) and represent the means of samples run in triplicate (±SD). Similar results were seen in two subsequent assays. (B) Trypsin pretreatment of S protein inactivates infectivity. HIV-luc(SARS S) infection of 293T/ACE2 cells was assessed as luciferase activity, presented as a percentage of no-trypsin control (≈40,000 RLU). The results represent the means of samples run in triplicate (±SD). (C) Trypsin treatment bypasses ammonium chloride inhibition of SARS-CoV. Mock- (Center) or 25 mM ammonium chloride-pretreated (Right) Vero E6 cells were spin-infected with replication-competent SARS-CoV at a multiplicity of infection of 0.5 and incubated with either DMEM (Upper) or DMEM containing TPCK-trypsin (Lower). After 48 h, the cells were immunostained for S protein.
Fig. 2.
Fig. 2.
Protease-inhibitor sensitivity. (A) Leupeptin inhibits S protein-mediated infection. The 293T cells were preincubated with leupeptin and challenged with HIV-luc SARS S (solid line, ♦), VSV-G (dashed line, ▪), or MLV-Ampho (dotted line, ▴). The results are presented as a percentage of infection of untreated cells (≈3,000 RLU) for each envelope) and represent the means of samples run in triplicate (±SD). Similar results were seen in two subsequent assays. (B) Leupeptin inhibits replication-competent SARS-CoV infection. Cells were either preincubated with leupeptin for 1 h and then exposed to virus for 3 h in the continued presence of leupeptin (solid line) or exposed to virus for 3 h and incubated for an additional 4 h with leupeptin (dashed line). At 3 days postexposure, the supernatant was analyzed for nucleoprotein by ELISA. The results are expressed as OD and represent the means of samples run in triplicate (±SD). Similar results were seen in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium cytoxicity assay. (C) Trypsin treatment bypasses leupeptin inhibition of live SARS-CoV. Mock- (Center) or 500 μg/ml leupeptin-pretreated (Right) Vero E6 cells were spin-infected with replication-competent SARS-CoV at a multiplicity of infection of 0.5 and incubated with either DMEM (Upper) or DMEM containing TPCK-trypsin (Lower). After 48 h, the cells were immunostained for S protein. (D) E64c blocks SARS-CoV S protein-mediated entry. The 293T cells were preincubated with E64c (solid lines) or aprotinin (dashed lines) and challenged with HIV-luc SARS S (black lines) or VSV-G (gray lines). The results are presented as a percentage of infection of untreated cells (≈1,500 RLU for VSV-G and 6,000 RLU for SARS S) and represent the means of samples run in triplicate (±SD). Similar results were seen in two additional experiments. (E) Z-lll-FMK inhibits S protein-mediated infection. Vero E6 cells were preincubated with Z-lll-FMK (solid lines) or CA-074 (dashed lines) and then challenged with HIV-luc SARS S (black lines) or VSV-G (gray lines). The results are presented as a percentage of infection of untreated cells (≈15,000 RLU for VSV-G and 20,000 RLU for SARS S) and represent the means of samples run in triplicate (±SD). Similar results were seen on 293T and 293T/ACE2 cells.
Fig. 3.
Fig. 3.
Cathepsin-L-specific inhibitor blocks infection. (A) MDL28170 inhibits CTSL activity with an IC50 of 2.5 nM. A 1,000-compound library was screened for inhibitors of CTSL activity (Inset, bottom left). MDL28170 (Inset, top right) was found to be a potent inhibitor. The compound library was screened against several other cathepsins, including CTSB, with no hits. The activity of MDL28170 was confirmed in an in vitro CTSL-cleavage assay (inhibition curve). (B) MDL28170 inhibits S protein-mediated infection. The 293T cells were preincubated with MDL28170 and challenged with HIV-luc SARS S (solid line) or VSV-G (dashed line). The results are presented as a percentage of infection of untreated cells (≈100,000 RLU for VSV-G and 20,000 RLU for SARS S) and represent the means of samples run in triplicate (±SD). Similar results were seen on Vero E6 and 293T/ACE2 cells.
Fig. 4.
Fig. 4.
S protein-mediated intervirion fusion. (A) Intervirion fusion requires ACE2 and S protein. Bald or ACE2 particles encoding luciferase (x axis) were incubated with particles encoding GFP (SARS S and ASLV-A envelope, gray bars; SARS S alone, black bars; or ASLV-A envelope alone, white bars). Virions were mixed and used to infect HeLa/Tva cells that had been pretreated with medium in the presence and absence of leupeptin (Leu) (20 μg/ml). Intervirion fusion was measured as luciferase activity 48 h postinfection. Results represent the means of samples run in triplicate (±SD). (B) Trypsin cleavage promotes fusion mediated by S protein. Intervirion fusion between HIV-luc(ACE2) and HIV-gfp(SARS S/ASLV-A) treated with TPCK-trypsin (10 μg/ml) for 10 min at 25°C or pulsed at pH 5.0 was quantified by luciferase activity 48 h postinfection of HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in triplicate (±SD). Mixtures of HIV-gfp(SARS S), HIV-gfp(ASLV-A), and HIV-luc(ACE2) could not be activated by trypsin cleavage, suggesting that S and ASLV-A envelope are required to be incorporated into the same particle in order for transduction of _target cells by fused particles. (C) Receptor interactions at elevated temperature are required before trypsin cleavage. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and incubated at 4°C to allow binding. Samples were then incubated at the noted temperatures. TPCK-trypsin digestion was carried out at 4°C for 15 min. The results represent the means of samples run in quadruplicate (±SD). Similar results were observed in two additional experiments. Temp., temperature. (D) CTSL enhances intervirion fusion. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and incubated for 10 min at 25°C with preactivated CTSB (at pH 5.0), CTSL (at pH 6.0), CTSL buffer alone (at pH 6.0), or TPCK-trypsin (at pH 7.0). The mixed virus was used to infect HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in quadruplicate (±SD). Similar results were observed in two subsequent experiments. (E) Acidic conditions are required for CTSL-mediated S protein activation. HIV-luc(ACE2) and HIV-GFP(SARS S/ASLV-A) particles were mixed and adjusted to various pHs and CTSL was added. After neutralization of acid conditions, the mixed virus was used to infect HeLa/Tva cells pretreated with leupeptin. The results represent the means of samples run in quadruplicate (±SD). Tryp, trypsin. Similar results were observed in an additional experiment.
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