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

Ilian Radichev

Sergey A. Shiryaev

Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA

Correspondence
Alex Y. Strongin
strongin{at}burnham.org

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 G²² residue and the negatively charged patch D³²DD³⁴ of NS2B were part of an important configuration required for NS2B–NS3pro activity. Our experimental data confirmed that G²² and D³²DD³⁴ 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.

These authors contributed equally to this work.

The sequences of the mutant primers are available with the online version of this paper.