Fig 5. Role and virus specificity of HCV NS3_N domain as NS2 protease cofactor.

(A) Schematic representation of the chimeric NS2^HCV-NS3_N^hepaci-ST precursors. Precursors spanning NS2 from HCV-JFH1 and NS3_N from NPHV, BHV, GHV, RHV or GBV-B (^hepaci) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). The sequence alignment of NS3 N-terminal sequences from the various hepaciviruses is depicted in the blown-up scheme with respect to HCV corresponding sequence, where identical residues are indicated by dots. (B) Extracts from cells transfected with pCMV/NS2^HCV(wt)-NS3_N^hepaci-ST or pCMV/NS2^HCV(CA)-NS3_N^hepaci-ST DNAs encoding NS2 protease with either native (wt) or mutated (CA) catalytic triad, respectively, were probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. The decreasing overall sequence similarity of hepacivirus NS3_N with respect to HCV NS3_N is represented by a grey triangle.