Expression and Characterization of the HCV NS2 Protease

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 –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 –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 ).