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Journal of Virology, December 2004, p. 13708-13716, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.13708-13716.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation

Pornwaratt Niyomrattanakit, Pakorn Winoyanuwattikun, Santad Chanprapaph, Chanan Angsuthanasombat, Sakol Panyim, and Gerd Katzenmeier*

Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Thailand

Received 26 February 2004/ Accepted 8 August 2004

Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed {Phi}x3{Phi} motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a kcat/Km value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced kcat values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.

* Corresponding author. Mailing address: Laboratory of Molecular Virology, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon 4 Rd., Nakornpathom 73170, Thailand. Phone: (662)-800-3624-1237. Fax: (662)-441-9906. E-mail: frkgz{at}mahidol.ac.th.

Journal of Virology, December 2004, p. 13708-13716, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.13708-13716.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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