NS2 proteases from hepatitis C virus and related hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities
Fig 2
Hepacivirus NS2 autoprotease activity.
(A) Schematic representation of the hepacivirus NS2-NS3N-ST precursors. Precursors spanning NS2 and NS3 N-terminal domains (NS3N) were expressed downstream of a heterologous signal peptide (sp) and C-terminally fused to Strep-tag (ST). NS3N comprises two amino acid residues (H, D; thin solid lines) of putative or established NS3 protease catalytic triads, whereas the third residue (Ser) was mutated into Ala (SA, thin dotted line). NS2(wt)-NS3N–ST precursors contain the native residues of the putative or established NS2 protease catalytic triads (H, E/D and C, thick solid lines). NS2(CA)-NS3N-ST precursors bear an Ala substitution of the catalytic Cys residue (CA, thick dotted line) in NS2. (B) Proteins extracted from cells transfected with pCMV/NS2(wt)-NS3N-ST or pCMV/NS2(CA)-NS3N-ST DNAs, allowing expression of native or mutated NS2-NS3N-ST precursors of the indicated viruses, respectively, were separated by SDS-PAGE and probed with anti-ST antibodies. Uncleaved precursors and cleaved products are indicated by closed and open arrowheads, respectively.