Publication: Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation
1
Issued Date
2004-12-01
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ISSN
0022538X
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2-s2.0-10044278380
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Virology. Vol.78, No.24 (2004), 13708-13716
Suggested Citation
Pornwaratt Niyomrattanakit, Pakorn Winoyanuwattikun, Santad Chanprapaph, Chanan Angsuthanasombat, Sakol Panyim, Gerd Katzenmeier Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. Journal of Virology. Vol.78, No.24 (2004), 13708-13716. doi:10.1128/JVI.78.24.13708-13716.2004 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/21334
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Title
Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation
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Abstract
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 ΦX 3Φ 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/K m 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.