Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactorNS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease d omain (NS3pro) that requires the conserved hydrophilic domain of NS2B for p rotease activity in cleavage of the polyprotein precursor at sites followin g two basic amino acids. In this study, we report the expression of the NS2 B-NS3pro precursor in Escherichia cole as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni2+ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified activ e protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluo rogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly con served Gly residue at P3 position was 3-fold more active as a substrate tha n a Gin residue at this position. The cleavage of a chromogenic substrate w ith 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.