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J. Biol. Chem., Vol. 280, Issue 31, 28766-28774, August 5, 2005

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Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries^*

Jun Li, Siew Pheng Lim¶, David Beer¶, Viral Patel¶, Daying Wen¶, Christine Tumanut, David C. Tully, Jennifer A. Williams, Jan Jiricek, John P. Priestle||, Jennifer L. Harris**, and Subhash G. Vasudevan¶

From the Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, the ^¶Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670, and the ^||Novartis Institute for Biomedical Research, CH-4002 Basel, Switzerland

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.

Received for publication, January 18, 2005 , and in revised form, May 26, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables I ("Steady-state kinetic parameters of DEN2 CF40-Gly-NS3pro185 using fluorogenic hexapeptides with natural dengue cleavage sites") and II ("P4-P1 preference of dengue 1 NS3 protease").

Both contributed equally to this work.

^** To whom correspondence may be addressed. E-mail: jharris{at}gnf.org.

To whom correspondence may be addressed. E-mail: subhash.vasudevan{at}group.novartis.com.

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