Abstract
This study was conducted to examine the effect of cystatin D, a newly discovered salivary cysteine protease inhibitor, on human coronavirus replication. When MRC‐5, human diploid lung cells, were incubated with dilutions of recombinant human cystatin D from 0.65–10 μM for 1 h prior to, during and after infection with coronavirus OC43 and 229e strains, a decrease in virus yield was observed resulting in an IC50 of 0.8 μM for both virus strains. This dose is within the normal concentration range of cystatin D, 0.12–1.9 μM found in saliva. When a single dose, 2.5 μM, was applied, cystatin inhibition of release of virus progeny was not overcome until three days post infection whereas inhibition by leupeptin, a serine and cysteine protease inhibitor, was completely abrogated by two days. When cellular toxicity was measured by 3H‐thymidine uptake, cystatin D did not markedly affect cell proliferation below a 10 μM dose. The results demonstrate that cystatin D is a potent inhibitor of coronavirus replication.
Keywords: cystatins, human coronavirus, inhibition
References
- 1. Abrahamson M., Barrett, AJ , Salvenson G., Grubb A. Isolation of six cysteine proteinase inhibitors from human urine. Their physiologic and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 1986: 261: 11282–11289. [PubMed] [Google Scholar]
- 2. Appleyard G. Tisdale M. Inhibition of the growth of human coronavirus 229e by leupeptin. J Gen Virol 1985: 66: 363–366. [DOI] [PubMed] [Google Scholar]
- 3. Balbin M., Hall A., Grubb A., Mason RW, Lopez‐Otin C., Abrahamson M. Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases. J Biol Chem 1994: 269: 23156–23162. [PubMed] [Google Scholar]
- 4. Bjorck L., Grubb A., Kjellen L. Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus. J Virol 1990: 64: 941–943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Collins A., Grubb A. Inhibitory effects of recombinant human cystatin C on human coronaviruses. Antimicrob Agents Chemother 1991: 35: 2444–2446. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Freije JP, Balbin M., Abrahamson M. et al. Human cystatin D. cDNA cloning, characterization of the Escherichia coli expressed inhibitor and identification of the native protein in saliva. J Biol Chem 1993: 268: 15737–15744. [PubMed] [Google Scholar]
- 7. Gu M., Haraszthy G., Collins AR, Bergey J. Identification of salivary proteins inhibiting herpes simplex virus 1 replication. Oral Microbiol Immunol 1995: 10: 54–59. [DOI] [PubMed] [Google Scholar]
- 8. Herold J., Raabe T., Schelle‐Prinz B., Siddell SG. Nucleotide sequence of the human coronavirus 229e RNA polymerase locus. Virology 1993: 195: 680–691. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Katoh I., Yasunaga T., Ikawa Y., Yohinaka Y. Inhibition of retroviral protease activity by an aspartyl proteinase inhibitor. Nature 1987: 329: 654–656. [DOI] [PubMed] [Google Scholar]
- 10. Korant B., Brzin J., Turk V. Cystatin, a protein inhibitor of cysteine proteases alters viral protein cleavages in infected human cells. Biochem Biophys Res Commun 1985: 127: 1072–1076. [DOI] [PubMed] [Google Scholar]
- 11. Myint SH. Human coronaviruses; a brief review. Rev Med Virol 1994: 4: 35–46. [Google Scholar]
- 12. Sadoul R., Fernandez PA, Quiquerez AL et al. Involvement of the proteasome in the programmed cell death of NGF‐deprived sympathetic neurons. EMBO J. 1996: 15: 3845–3852. [PMC free article] [PubMed] [Google Scholar]
- 13. Taugner R., Buhrle CP, Nobiling R., Kirschke H. Coexistence of renin and cathepsin B in epitheloid cell secretory granules. Histochemistry 1995: 83: 103–108. [DOI] [PubMed] [Google Scholar]
- 14. Schroder E., Phillips C., Garman E., Harlos K., Crawford C. X‐ray crystallographic structure of a papain‐leupeptin complex. FEBS Lett 1993: 315: 38–42. [DOI] [PubMed] [Google Scholar]