Fig 3. NS2 N-terminal domain requirement for proteolytic activity.
(A) Schematic representation of the hepacivirus ΔN(NS2)-NS3N-ST truncated precursors, which are derived from NS2-NS3N-ST polypeptides (see Fig 1 legend) and lack the hydrophobic N-terminal region of NS2. (B) Proteins extracted from cells transfected with pCMV/ΔN(NS2)(wt)-NS3N-ST or pCMV/ΔN(NS2)(CA)-NS3N-ST DNAs, allowing the transient expression of native (wt) or mutated (CA) ΔN(NS2)-NS3N-ST truncated precursors (ΔN) of the indicated viruses, respectively, were separated by SDS-PAGE and immunodetected with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively. (C-D) HCV and GBV-B full-length (HCV, GBV-B) or truncated (ΔN HCV, ΔN GBV-B) precursors were expressed in a wheat germ cell-free expression system in the absence (C) or in the presence (D) of detergent MNG-3. Cell-free expression samples were affinity purified using Strep-Tactin beads, separated by SDS-PAGE and probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively.