FIGURE 1.

Constructs of the NS2-NS3 proteinase-helicase of WNV. A, the structure of the WNV polyprotein precursor (GenBank™ entry YP_001527877). The numbering starts from the N-terminal methionine. The 48-residue central portion of NS2B (residues 49–96 of the NS2B sequence; see supplemental Fig. S1) was linked with the 184-residue NS3pro sequence (residues 1505–1689 of the polyprotein precursor sequence) via a GGGGSGGGG linker. The Ala residue substituted for the essential His⁵¹ in the H51A inert mutant. The Ala substituted for Lys⁴⁸ in the autolytic site-deficient K48A mutant. The K48A and H51A NS2B-NS3pro constructs were linked to the NS3hel sequence (residues 171–625 of the NS3 sequence) to restore the natural sequence of NS3 and to obtain the full-length NS2B-NS3pro-hel K48A (autolytic site-deficient) and NS2B-NS3pro-hel H51A (proteolytically inert) mutant constructs. To construct NS3pro-hel, the NS2B portion was truncated in the wild-type NS2B-NS3pro-hel construct. The N-terminal Met¹⁷¹ was used for the translation initiation. The Leu⁸⁵ NS3pro-hel species we identified represents the proteolytic fragment of the NS3pro-hel construct because of the aberrant proteolysis of the latter by E. coli proteinases. The constructs were C-terminally tagged with a His₆ tag. B, the N-terminal amino acid sequence of NS2B-NS3pro-hel. The positions of the K48A and H51A mutants are indicated by the residue number and the vertical arrow. The N-terminal Leu⁸⁵ of the 60-kDa proteolytic fragments of the NS2B-NS3pro-hel that we identified is numbered and underlined. The linker sequence is italicized. The initiation methionine (not shown) was added to the N terminus of the NS3pro-hel construct. C, model of membrane-bound WNV NS2B-NS3pro-hel. The N- and C-terminal lobes of NS3pro are shown in green and cyan, and the NS2B cofactor is in magenta. The NS3pro Asp-His-Ser catalytic triad is shown as black dots. The relative positions of the N and C termini of the NS2B cofactor are arbitrary but consistent with the lengths of the linker regions (shown as dotted lines). Because the junction region between NS2B and NS3pro can be auto-proteolyzed, the C-terminal region of NS2B is placed into the NS3pro active site. Ser⁴⁹ and Lys⁹⁶ are the N- and C-terminal residues, respectively, of the NS2B cofactor used in our constructs.