Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro.

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

Crystallization, characterization and measurement of MAD data on crystals of dengue virus NS3 serine protease complexed with mung-bean Bowman-Birk inhibitor.

Crystallization and preliminary characterization of the essential dengue virus NS3 serine protease complexed with a Bowman-Birk-type inhibitor from mung beans are reported. As the structure proved resistant to solution by molecular replacement and multiple isomorphous replacement methods, multi-wavelength anomalous diffraction data at the LIII edge of a holmium derivative have been measured. Promising Bijvoet and dispersive signals which are largely consistent with expected values have been extracted from the data. The structure, when determined, will provide a structural basis for the design, synthesis and evaluation of inhibitors of the protease for chemotherapy of dengue infections.

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.

Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects.

The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases.

Cotranslational membrane insertion of the serine proteinase precursor NS2B-NS3(Pro) of dengue virus type 2 is required for efficient in vitro processing and is mediated through the hydrophobic regions of NS2B.

Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in vitro processing system, we examined the cis and trans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 site in vitro, and the protease activity was associated with the membrane fraction. To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitro transcription/translation system (TnT). The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner. NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were membrane-associated. Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 site in vitro in the absence of microsomal membranes.