Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virusencoded 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 ⌽x 3 ⌽ 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 k cat /K m value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k cat 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.Infection by dengue viruses is now widely recognized as a major public health concern, with more than 1 million cases of dengue hemorrhagic fever per year and case fatality rates ranging from 1 to 10% (23). There are four serotypes of dengue virus, which cause dengue hemorrhagic fever and dengue shock syndrome (21,22). Dengue viruses, members of the Flaviviridae family, are small, enveloped, positive-stranded RNA viruses which are transmitted by Aedes mosquitoes (7). At present, neither a commercial vaccine nor a causative treatment is available for the prevention or cure of acute dengue virus diseases.The genomic RNA of dengue virus serotype 2 contains 10,723 nucleotides and encodes a large polyprotein precursor of 3,391 amino acid residues which consists of three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (26). The polyprotein is co-and posttranslationally processed by proteases of the host cell and the virus-encoded two-component protease NS2B-NS3 to generate individual viral proteins (11,18). Optimal activity of the NS3 serine protease (flavivirin, EC 3.4.21.91) is an essential requirement for maturation of the virus, and inhibition of this enzyme offers the prospect of an effective antiviral chemotherapy for severe cases of dengue hemorrhagic fever and dengue shock syndrome (for review see references 6, 39, and 41 and...