Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Abstract

ABSTRACT The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1′-P2′ positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1′ or P2′. Structure-based modeling suggests that Arg 76 and Pro 131 -Thr 132 limit the P1′-P2′ subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu 76 and Lys 131 -Pro 132 widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3′-P4′ positions were fixed and the P1′ and P2′ positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.