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...

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.

The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.

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