Steady-State Cleavage Kinetics for Dengue Virus Type 2 Ns2b-Ns3(Pro) Serine Protease With Synthetic Peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B / NS3, NS3 / NS4A and NS4B / NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for Km, kcat and kcat / Km were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B / NS3 site. A marked difference in cleavage efficiency was found for the NS2B / NS3 substrate and the remaining 3 peptides based on the NS2A / NS2B, NS3 / NS4A and NS4A / NS5 cleavage sites.