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Journal of Virology, May 2007, p. 4501-4509, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02719-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Sergey A. Shiryaev,1 Boris I. Ratnikov,1 Alexander E. Aleshin,1 Igor A. Kozlov,2 Nicholas A. Nelson,2 Michal Lebl,2 Jeffrey W. Smith,1 Robert C. Liddington,1 and Alex Y. Strongin1*

Burnham Institute for Medical Research, La Jolla, California,1 Illumina, Inc., San Diego, California2

Received 9 December 2006/ Accepted 19 January 2007

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 Arg76 and Pro131-Thr132 limit the P1'-P2' subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu76 and Lys131-Pro132 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.

* Corresponding author. Mailing address: The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA. Phone: (858) 713-6271. Fax: (858) 713-9925. E-mail: strongin{at}burnham.org

{triangledown} Published ahead of print on 14 February 2007.

Journal of Virology, May 2007, p. 4501-4509, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02719-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Remacle, A. G., Shiryaev, S. A., Oh, E.-S., Cieplak, P., Srinivasan, A., Wei, G., Liddington, R. C., Ratnikov, B. I., Parent, A., Desjardins, R., Day, R., Smith, J. W., Lebl, M., Strongin, A. Y. (2008). Substrate Cleavage Analysis of Furin and Related Proprotein Convertases: A COMPARATIVE STUDY. J. Biol. Chem. 283: 20897-20906 [Abstract] [Full Text]  
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