Dengue viruses, members of the Flaviviridae are recognized as the causative agent or one of the most important mosquito-borne viral diseases of humans affecting 100 million people annually. Of these, more than 250,000 cases involve life-threatening dengue hemorrhagic fever/dengue shock syndrome with no effective chemotherapy or vaccine currently available. One of the long term goals of this study is to dissect the biochemical mechanism(s) by which dengue virus replicates in the permissive host cells. Processing of the viral polyprotein precursor in host cells is carried out by the two-component viral protease, NS2B/NS3, which plays a pivotal role in the virus life cycle. A hydrophilic region of 40 amino acids of NS2B, SN2B(H), serves as an activator of the catalytic subunit of the NS3 serine protease domain (NS3-pro). In addition to the protease activity, NS3 has RNA-stimulated NTPase, RNA helicase, and 5' RNA triphosphatase activities. The structures of NS3-pro and the NS3-pro/mung bean inhibitor (MbBBI) complex have been determined. Kinetic characterization of NS3-pro and NS2B(H)/NS3-pro have also been carried out. The objectiveof the research proposed in this application is to dissect the molecular mechanism of activation of dengue virus serine protease using structural and biochemical approaches. This research objective will be accomplished by the completion of the following Specific Aims: (1)To understand the biochemical mechanism of the viral protease, kinetic parameters of both NS3-pro and NS2B(H)/NS3-pro will be determined using fluorogenic peptide substrates and protease inhibitors. (2) To elucidate the mechanism of activation of NS3-pro by NS2B(H), structures of ternary complexes of NS2B(H)/NS3-pro and a suitable serine protease inhibitors will be determined. (3) Site specific mutations suggested by the structure of NS3-pro/MbBBI will be made and characterized functionally and structurally. The work to be carried out will result in laying the necessary structural foundation for structure- assisted design of specific inhibitors of the dengue virus protease. It will also permit a structural definition of alternate targets for inhibition of the protease and their detailed evaluation.
