Dengue virus, a member of the Flaviviridae family, is a small, spherical, enveloped, positive single strand RNA virus that is transmitted to humans by mosquitoes of the species Stegomyia aegypti (formerly Aedes). All 4 serotypes of the virus (DEN-1, 2, 3 and 4) can cause a spectrum of clinical symptoms including mild dengue fever (DF) and more severe forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [1, 2]. An increase of geographical spread, incidence and severity of diseases over the past decade has now stimulated intensive efforts to develop effective antiviral therapeutics which are eventually useful for the prevention and cure of dengue virus infections. The development of small molecule drugs directed at inhibition of replication and maturation of the virus is now considered as promising route for the treatment of acute dengue diseases [for review see[3–5]and references herein].
The dengue virus NS3 protease, a member of the flavivirin enzyme family (EC 220.127.116.11), is located in the N-terminal 184 residues of the multifunctional 69 kDa NS3 protein and contains a functional catalytic triad consisting of H51, D75 and S135 (in DEN-2) . In addition to the serine protease, the NS3 protein contains enzymatic activities of a nucleoside triphosphatase, a 5' - RNA triphosphatase (RTPase) and a RNA - stimulated RNA helicase [7, 8]. The NS3 protease catalyses the post-translational cleavage of the viral polyprotein precursor in the non-structural region at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites and at additional sites within the viral capsid protein, NS2A, NS4A and within a C-terminal region of NS3 itself [9–13]. The overall conformation of the dengue virus NS3 protease displays the β-barrel conformation typical for serine proteases, although the viral enzyme appears to possess higher compactness with short or absent loop structures and a relatively shallow substrate binding site .
The presence of a small hydrophilic core segment of approximately 40 residues, commonly designated NS2B(H), within the small 14 kDa NS2B cofactor is required for optimal activity of the NS3 protease [15–17]. Proteolytic autoprocessing at the NS2B/NS3 site generates a non-covalent adduct between NS2B(H) and NS3 which is catalytically active with substrates supplied in trans cleavage reactions .
Detailed substrate specificity studies have established that the cleavage junctions in the viral polyprotein consist of pairs of dibasic amino acids such as RR, RK and KR at the P1 and P2 positions. Small, non-branched amino acids such as S are preferred at the P1' position of the dengue virus cleavage site, whereas the preferred P1' residue of the WNV NS3 protease is G [19–21].
Theoretical molecular interactions between the active site of the NS3 protease and the peptide substrate were largely consistent with data obtained from substrate profiling studies . Crystallographic studies of flaviviral proteases including the West Nile Virus (WNV) and dengue virus in complex with a partial NS2B cofactor and substrate-mimetic inhibitors such as aprotonin have provided evidence for major structural reorganizations of the active site pockets caused by insertion of a β-barrel of the NS2B cofactor and an "induced fit" mechanism of catalysis in the presence of authentic protein substrates . Based on a homology-modelled structure of the WNV NS3 protease, residues within the S1 and S2 pockets critical for enzyme-substrate interaction were identified by analysis of catalytic activity of mutant proteases with a synthetic peptide substrate . Structural data obtained recently for a WNV NS2B-NS3pro protease in complex with a substrate-based tripeptide inhibitor have revealed a catalytically competent oxyanion binding site formed by two residues, G133 and S135, and substitution of the active-site nucleophile serine by alanine does not result in a disruption of the oxyanion conformation . It is noteworthy that also in the presence of ligands without a P1' residue the active conformation of the oxyanion hole is adopted by the viral protease.
A high conservation of sequences within the faviviral proteases suggests that specificity characteristics found for the WNV protease could also be of relevance for the closely related dengue virus NS3 protease. Despite their overall similarities, the NS3 proteases from dengue virus and WNV exhibit different substrate specificities, suggesting a distinct organization of their respective active site conformations .
In analogy to procedures previously described for the enzyme from WNV, we have identified key residues for substrate binding and catalysis of the dengue virus NS3 protease by alanine substitution mutagenesis and assay of the recombinant mutant enzymes with a synthetic model substrate. In fact, an earlier study has described extensive mutagenesis within the dengue virus NS3 protease for ultraconserved residues among flaviviral proteases and these residues were putatively involved in catalysis or substrate binding . However, activity of the mutant proteases was assayed by SDS-PAGE analysis of autoproteolytic cleavage of the NS2B-NS3 precursor in vivo. Although this approach yielded semiquantitative data for activity of the mutant enzymes, it did not provide precise numerical values for the kinetic activity of the mutant proteases with substrates supplied for trans cleavage reactions. Moreover, a number of residues such as L115, S163 and I165 have not been included in that investigation as their possible role for enzyme activity was suggested later by data from structural experiments [14, 23]. Therefore, the changes in catalytic efficiency which we have observed in the context of amino acid exchanges could contribute to a refined model of substrate specificity and active site conformation for the dengue virus NS3 protease.