Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B-NS3 proteinase

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B - NS3pro structure, we hypothesized that the G(22) residue and the negatively charged patch (DDD34)-D-32 of NS2B were part of an important configuration required for NS2B - NS3pro activity. Our experimental data confirmed that G(22) and (DDD34)-D-32 substitution for S and AAA, respectively, inactivated NS2B - NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B - NS3pro. Because of the significant level of homology in flaviviral NS2B - NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.