Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry - PubMed (original) (raw)

Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry

Graham Simmons et al. Proc Natl Acad Sci U S A. 2005.

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.

PubMed Disclaimer

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.

Similar articles

Cited by

References

    1. Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P., Penaranda, S., Bankamp, B., Maher, K., Chen, M. H., et al. (2003) Science 300, 1394-1399. - PubMed
    1. Guan, Y., Zheng, B. J., He, Y. Q., Liu, X. L., Zhuang, Z. X., Cheung, C. L., Luo, S. W., Li, P. H., Zhang, L. J., Guan, Y. J., et al. (2003) Science 302, 276-278. - PubMed
    1. Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454. - PMC - PubMed
    1. Simmons, G., Reeves, J. D., Rennekamp, A. J., Amberg, S. M., Piefer, A. J. & Bates, P. (2004) Proc. Natl. Acad. Sci. USA 101, 4240-4245. - PMC - PubMed
    1. Yang, Z. Y., Huang, Y., Ganesh, L., Leung, K., Kong, W. P., Schwartz, O., Subbarao, K. & Nabel, G. J. (2004) J. Virol. 78, 5642-5650. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources