Fig 4. Dimerization of HCV, NPHV and GBV-B NS2 proteases.

(A) Schematic representation of NS2 protease dimer formation upon co-expression of mutated precursors. The co-expression of NS2-NS3_N precursors (CA ST and HA V5 shown as examples) bearing an Ala substitution of either Cys (CA, red code) or His (HA, green code) catalytic residue and C-terminally fused to Strep-tag (ST, black code) or V5 tag (V5, blue code), respectively, may lead to the formation of NS2-NS3_N dimers. Homo- and hetero-dimerizations of NS2 protease, represented according to the three-dimensional crystallographic structure of NS2 catalytic domain (PDB accession number 2HD0) [48], result in the formation of native (wt), single-mutant (HA or CA) or double-mutant (HA and CA) NS2 active sites, as symbolized by scissors, one or two cross signs, respectively. (B-D) Cells were transfected or co-transfected with one (lanes 1 to 6) or two (lanes 7 to 10) pCMV DNAs, allowing the transient expression of NS2-NS3_N precursors from HCV-JFH1 (B), NPHV (C) or GBV-B (D). Uncleaved precursors and cleaved products were immunodetected with anti-ST or anti-V5 antibodies (upper and lower images, respectively in each panel) following SDS-PAGE separation of transfected cell extracts and are indicated by closed and open arrowheads, respectively.