Expression and Characterization of the HCV NS2 Protease

  • Karen E. Reed
  • Charles M. Rice
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 19)

Abstract

Heterologous expression systems have been widely used to study hepatitis C virus (HCV) proteins in lieu of an efficient method for establishing HCV infections in cell culture. Studies of HCV polyprotein processing in both mammalian-cell-based and cell-free expression systems have shown that host signalase is responsible for cleavages between the structural proteins and at the N-terminus of NS2 (1, 2, 3, 4), whereas a chymotrypsin-like serine protease located in the N-terminal region of NS3 is responsible for most cleavages between the nonstructural proteins, including the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites (5, 6, 7, 8, 9, 10). However, two observations indicated that the 2/3 site is cleaved by a distinct virus-encoded protease, known as the NS2, NS2-3, or Cpro-2 protease: (1) mutation of the catalytic serine at amino acid (aa) 1165 of NS3 abolished cleavage at all sites in the nonstructural polyprotein except the 2/3 site, and (2) two mutations in NS2 located more than 30 residues from the NS2-NS3 junction abolished cleavage at the 2/3 site, but had little or no effect on downstream cleavages catalyzed by the serine protease (8,11). The minimal region required for cleavage at the 2/3 site has been mapped to aa 898 on the N-terminal side (8) and aa 1207 on the C-terminal side (11) (numbers refer to HCV 1a and 1b isolates). Although a single construct extending from aa 898 to 1207 has never been tested for proteolytic viability, cleavage with an efficiency similar to that of polypeptides containing full-length NS2 and NS3 has been demonstrated for truncated constructs extending from aa 898 to 1233 or 827 to 1207 (8,11).

Keywords

Codon Electrophoresis Serine Proline Polypeptide 

References

  1. 1.
    Hijikata, M., Kato, N, Ootsuyama, Y., Nakagawa, M., and Shimotohno, K. (1991) Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc. Natl Acad. Sci. USA 88, 5547–5551.PubMedCrossRefGoogle Scholar
  2. 2.
    Lin, C., Lindenbach, B. D., Prágai, B., McCourt, D. W., and Rice, C. M. (1994) Processing of the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol. 68, 5063–5073PubMedGoogle Scholar
  3. 3.
    Mizushima, H., Hijikata, H., Asabe, S.-I, Hirota, M., Kimura, K., and Shimotohno, K. (1994) Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 68, 6215–6222PubMedGoogle Scholar
  4. 4.
    Selby, M. J, Glazer, E., Masiarz, F., and Houghton, M. (1994) Complex processing and protein: protein interactions in the E2.NS2 region of HCV. Virology 204, 114–122.PubMedCrossRefGoogle Scholar
  5. 5.
    Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobsen, H. (1993) Nonstructural protein 3 of the hepatitis C virus encodes a senne-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67, 3835–3844PubMedGoogle Scholar
  6. 6.
    Eckart, M. R, Selby, M, Masiarz, F., Lee, C., Berger, K., Crawford, K., Kuo, C., Kuo, G., Houghton, M., and Choo, Q-L. (1993) The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Comm 192, 399–406PubMedCrossRefGoogle Scholar
  7. 7.
    Grakoui, A., McCourt, D. W, Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993) Characterization of the hepatitis C virus-encoded serine proteinase: determination of protemase-dependent polyprotein cleavage sites. J Virol 67,2832–2843.PubMedGoogle Scholar
  8. 8.
    Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., et al (1993) Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67,4665–4675PubMedGoogle Scholar
  9. 9.
    Manabe, S., Fuke, I., Tanishita, O., Kaji, C., Gomi, Y., Yoshida, S., Mori, S., et al (1994) Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3. Virology 198, 636–644PubMedCrossRefGoogle Scholar
  10. 10.
    Tomei, L., Failla, C., Santolini, E., De Francesco, R., and La Monica, N. (1993) NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol 67,4017–4026PubMedGoogle Scholar
  11. 11.
    Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993) A second hepatitis C virus-encoded proteinase. Proc Natl Acad. Sci USA 90, 10,583–10,587PubMedCrossRefGoogle Scholar
  12. 12.
    Hirowatari, Y., Hijikata, M., Tanji, Y., Nyunoya, H., Mizushima, H., Kimura, K., et al. (1993) Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector. Arch Virol. 133, 349–356PubMedCrossRefGoogle Scholar
  13. 13.
    Reed, K. E., Grakoui, A., and Rice, C. M. (1995) Hepatitis C virus-encoded NS2–3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage. J Virol 69,4127–4136PubMedGoogle Scholar
  14. 14.
    Santohni, E., Pacini, L., Fipaldini, C., Mighaccio, G., and La Monica, N. (1995) The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 69, 7461–7471.Google Scholar
  15. 15.
    Pieroni, L., Santohni, E., Fipaldini, C., Pacini, L., Mighaccio, G., and LaMonica, N. (1997) In vitro study of the NS2-3 protease of hepatitis C virus. J. Virol 71,6373–6380PubMedGoogle Scholar
  16. 16.
    Jiang, W. and Bond, J. S. (1992) Families of metalloendopeptidases and their relationships. FEBSLett 312, 110–114CrossRefGoogle Scholar
  17. 17.
    Fuerst, T. R., Niles, E. G., Studier, F W., and Moss, B. (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl. Acad Sci USA 83, 8122–8126PubMedCrossRefGoogle Scholar
  18. 18.
    Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and Fuerst, T. R. (1990) New mammalian expression vectors. Nature (London) 348, 91,92CrossRefGoogle Scholar
  19. 19.
    Pelham, H. R. B. and Jackson, R. J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J. Biochem 67, 247–256PubMedCrossRefGoogle Scholar
  20. 20.
    Grakoui, A., Wychowski, C., Lin, C., Feinstone, S. M., and Rice, C. M. (1993) Expression and identification of hepatitis C virus polyprotein cleavage products. J. Virol 67, 1385–1395.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1998

Authors and Affiliations

  • Karen E. Reed
    • 1
  • Charles M. Rice
    • 1
  1. 1.Department of Molecular MicrobiologyWashington University School of MedicineSt. Louis

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