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. 1996 Oct;70(10):6694–6700. doi: 10.1128/jvi.70.10.6694-6700.1996

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

C Steinkühler 1, A Urbani 1, L Tomei 1, G Biasiol 1, M Sardana 1, E Bianchi 1, A Pessi 1, R De Francesco 1
PMCID: PMC190711  PMID: 8794305

Abstract

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.

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Selected References

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  1. Alter H. J. To C or not to C: these are the questions. Blood. 1995 Apr 1;85(7):1681–1695. [PubMed] [Google Scholar]
  2. Arias C. F., Preugschat F., Strauss J. H. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology. 1993 Apr;193(2):888–899. doi: 10.1006/viro.1993.1198. [DOI] [PubMed] [Google Scholar]
  3. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Kinetic and structural analyses of hepatitis C virus polyprotein processing. J Virol. 1994 Aug;68(8):5045–5055. doi: 10.1128/jvi.68.8.5045-5055.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartenschlager R., Ahlborn-Laake L., Mous J., Jacobsen H. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol. 1993 Jul;67(7):3835–3844. doi: 10.1128/jvi.67.7.3835-3844.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chambers T. J., Grakoui A., Rice C. M. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J Virol. 1991 Nov;65(11):6042–6050. doi: 10.1128/jvi.65.11.6042-6050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chien D. Y., Choo Q. L., Tabrizi A., Kuo C., McFarland J., Berger K., Lee C., Shuster J. R., Nguyen T., Moyer D. L. Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10011–10015. doi: 10.1073/pnas.89.21.10011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  8. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. D'Souza E. D., Grace K., Sangar D. V., Rowlands D. J., Clarke B. E. In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease. J Gen Virol. 1995 Jul;76(Pt 7):1729–1736. doi: 10.1099/0022-1317-76-7-1729. [DOI] [PubMed] [Google Scholar]
  10. Darke P. L., Chen E., Hall D. L., Sardana M. K., Veloski C. A., LaFemina R. L., Shafer J. A., Kuo L. C. Purification of active herpes simplex virus-1 protease expressed in Escherichia coli. J Biol Chem. 1994 Jul 22;269(29):18708–18711. [PubMed] [Google Scholar]
  11. DiIanni C. L., Mapelli C., Drier D. A., Tsao J., Natarajan S., Riexinger D., Festin S. M., Bolgar M., Yamanaka G., Weinheimer S. P. In vitro activity of the herpes simplex virus type 1 protease with peptide substrates. J Biol Chem. 1993 Dec 5;268(34):25449–25454. [PubMed] [Google Scholar]
  12. Eckart M. R., Selby M., Masiarz F., Lee C., Berger K., Crawford K., Kuo C., Kuo G., Houghton M., Choo Q. L. The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun. 1993 Apr 30;192(2):399–406. doi: 10.1006/bbrc.1993.1429. [DOI] [PubMed] [Google Scholar]
  13. Failla C., Tomei L., De Francesco R. An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A. J Virol. 1995 Mar;69(3):1769–1777. doi: 10.1128/jvi.69.3.1769-1777.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Failla C., Tomei L., De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol. 1994 Jun;68(6):3753–3760. doi: 10.1128/jvi.68.6.3753-3760.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10583–10587. doi: 10.1073/pnas.90.22.10583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hall D. L., Darke P. L. Activation of the herpes simplex virus type 1 protease. J Biol Chem. 1995 Sep 29;270(39):22697–22700. doi: 10.1074/jbc.270.39.22697. [DOI] [PubMed] [Google Scholar]
  20. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Shimotohno K. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5547–5551. doi: 10.1073/pnas.88.13.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hijikata M., Mizushima H., Akagi T., Mori S., Kakiuchi N., Kato N., Tanaka T., Kimura K., Shimotohno K. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol. 1993 Aug;67(8):4665–4675. doi: 10.1128/jvi.67.8.4665-4675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hijikata M., Mizushima H., Tanji Y., Komoda Y., Hirowatari Y., Akagi T., Kato N., Kimura K., Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirowatari Y., Hijikata M., Tanji Y., Nyunoya H., Mizushima H., Kimura K., Tanaka T., Kato N., Shimotohno K. Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector. Arch Virol. 1993;133(3-4):349–356. doi: 10.1007/BF01313774. [DOI] [PubMed] [Google Scholar]
  24. Jore J., De Geus B., Jackson R. J., Pouwels P. H., Enger-Valk B. E. Poliovirus protein 3CD is the active protease for processing of the precursor protein P1 in vitro. J Gen Virol. 1988 Jul;69(Pt 7):1627–1636. doi: 10.1099/0022-1317-69-7-1627. [DOI] [PubMed] [Google Scholar]
  25. Kakiuchi N., Hijikata M., Komoda Y., Tanji Y., Hirowatari Y., Shimotohno K. Bacterial expression and analysis of cleavage activity of HCV serine proteinase using recombinant and synthetic substrate. Biochem Biophys Res Commun. 1995 May 25;210(3):1059–1065. doi: 10.1006/bbrc.1995.1764. [DOI] [PubMed] [Google Scholar]
  26. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9524–9528. doi: 10.1073/pnas.87.24.9524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Komoda Y., Hijikata M., Tanji Y., Hirowatari Y., Mizushima H., Kimura K., Shimotohno K. Processing of hepatitis C viral polyprotein in Escherichia coli. Gene. 1994 Aug 5;145(2):221–226. doi: 10.1016/0378-1119(94)90009-4. [DOI] [PubMed] [Google Scholar]
  28. Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  29. Kuo G., Choo Q. L., Alter H. J., Gitnick G. L., Redeker A. G., Purcell R. H., Miyamura T., Dienstag J. L., Alter M. J., Stevens C. E. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science. 1989 Apr 21;244(4902):362–364. doi: 10.1126/science.2496467. [DOI] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Lin C., Amberg S. M., Chambers T. J., Rice C. M. Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J Virol. 1993 Apr;67(4):2327–2335. doi: 10.1128/jvi.67.4.2327-2335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lin C., Prágai B. M., Grakoui A., Xu J., Rice C. M. Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics. J Virol. 1994 Dec;68(12):8147–8157. doi: 10.1128/jvi.68.12.8147-8157.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lin C., Thomson J. A., Rice C. M. A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol. 1995 Jul;69(7):4373–4380. doi: 10.1128/jvi.69.7.4373-4380.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Overton H., McMillan D., Gillespie F., Mills J. Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays. J Gen Virol. 1995 Dec;76(Pt 12):3009–3019. doi: 10.1099/0022-1317-76-12-3009. [DOI] [PubMed] [Google Scholar]
  35. Pinko C., Margosiak S. A., Vanderpool D., Gutowski J. C., Condon B., Kan C. C. Single-chain recombinant human cytomegalovirus protease. Activity against its natural protein substrate and fluorogenic peptide substrates. J Biol Chem. 1995 Oct 6;270(40):23634–23640. doi: 10.1074/jbc.270.40.23634. [DOI] [PubMed] [Google Scholar]
  36. Pizzi E., Tramontano A., Tomei L., La Monica N., Failla C., Sardana M., Wood T., De Francesco R. Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):888–892. doi: 10.1073/pnas.91.3.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Santolini E., Migliaccio G., La Monica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol. 1994 Jun;68(6):3631–3641. doi: 10.1128/jvi.68.6.3631-3641.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sardana V. V., Wolfgang J. A., Veloski C. A., Long W. J., LeGrow K., Wolanski B., Emini E. A., LaFemina R. L. Peptide substrate cleavage specificity of the human cytomegalovirus protease. J Biol Chem. 1994 May 20;269(20):14337–14340. [PubMed] [Google Scholar]
  40. Satoh S., Tanji Y., Hijikata M., Kimura K., Shimotohno K. The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. J Virol. 1995 Jul;69(7):4255–4260. doi: 10.1128/jvi.69.7.4255-4260.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shimizu Y., Yamaji K., Masuho Y., Yokota T., Inoue H., Sudo K., Satoh S., Shimotohno K. Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. J Virol. 1996 Jan;70(1):127–132. doi: 10.1128/jvi.70.1.127-132.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shoji I., Suzuki T., Chieda S., Sato M., Harada T., Chiba T., Matsuura Y., Miyamura T. Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli. Hepatology. 1995 Dec;22(6):1648–1655. [PubMed] [Google Scholar]
  43. Steinkühler C., Tomei L., De Francesco R. In vitro activity of hepatitis C virus protease NS3 purified from recombinant Baculovirus-infected Sf9 cells. J Biol Chem. 1996 Mar 15;271(11):6367–6373. doi: 10.1074/jbc.271.11.6367. [DOI] [PubMed] [Google Scholar]
  44. Suzuki T., Sato M., Chieda S., Shoji I., Harada T., Yamakawa Y., Watabe S., Matsuura Y., Miyamura T. In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses. J Gen Virol. 1995 Dec;76(Pt 12):3021–3029. doi: 10.1099/0022-1317-76-12-3021. [DOI] [PubMed] [Google Scholar]
  45. Takamizawa A., Mori C., Fuke I., Manabe S., Murakami S., Fujita J., Onishi E., Andoh T., Yoshida I., Okayama H. Structure and organization of the hepatitis C virus genome isolated from human carriers. J Virol. 1991 Mar;65(3):1105–1113. doi: 10.1128/jvi.65.3.1105-1113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tomei L., Failla C., Santolini E., De Francesco R., La Monica N. NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol. 1993 Jul;67(7):4017–4026. doi: 10.1128/jvi.67.7.4017-4026.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Webster A., Hay R. T., Kemp G. The adenovirus protease is activated by a virus-coded disulphide-linked peptide. Cell. 1993 Jan 15;72(1):97–104. doi: 10.1016/0092-8674(93)90053-s. [DOI] [PubMed] [Google Scholar]
  48. Yamanaka G., DiIanni C. L., O'Boyle D. R., 2nd, Stevens J., Weinheimer S. P., Deckman I. C., Matusick-Kumar L., Colonno R. J. Stimulation of the herpes simplex virus type I protease by antichaeotrophic salts. J Biol Chem. 1995 Dec 15;270(50):30168–30172. doi: 10.1074/jbc.270.50.30168. [DOI] [PubMed] [Google Scholar]
  49. Ypma-Wong M. F., Dewalt P. G., Johnson V. H., Lamb J. G., Semler B. L. Protein 3CD is the major poliovirus proteinase responsible for cleavage of the P1 capsid precursor. Virology. 1988 Sep;166(1):265–270. doi: 10.1016/0042-6822(88)90172-9. [DOI] [PubMed] [Google Scholar]

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