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Originally published In Press as doi:10.1074/jbc.M107360200 on October 1, 2001

J. Biol. Chem., Vol. 276, Issue 49, 45762-45771, December 7, 2001
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Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors*

Donmienne LeungDagger , Kate Schroder§, Helen White§, Ning-Xia Fang§, Martin J. StoermerDagger , Giovanni AbbenanteDagger , Jennifer L. MartinDagger , Paul R. Young§, and David P. FairlieDagger

From the Dagger  Centre for Drug Design and Development, Institute for Molecular Bioscience and § Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mM, 1 mM CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

* This work was supported in part by the National Health and Medical Research Council of Australia and the Australian Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 61-7-33651268; Fax: 61-7-33651990; E-mail: d.fairlie@imb.uq.edu.au.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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