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)


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).


Codon Electrophoresis Serine Proline Polypeptide 


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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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