The overall aim of this proposal is to elucidate the role of the dengue viral antigens NS3 and NS5 in protein processing and genome replication. Dengue is a human disease caused by the mosquito-borne dengue virus.Apart from dengue fever, a more severe and fatal form of the disease occurs in children called dengue hemorrhagic fever, or the dengue shock syndrome. A potential problem associated with the strategy for the development of monovalent dengue vaccines is that individuals infected with one serotype are fully susceptible to infection by others. This secondary infection may result in the severe and fatal form of the disease. One approach to control the virus infection is to understand the replication strategy of the virus so that an antiviral agent can be developed. The viral antigens, NS3 and NS5, are useful targets for such studies, because they are highly conserved flavivirus nonstructural proteins, which have been implicated in protein processing and viral replication. NS3 was shown to contain a serine protease domain within the first 180 amino acids. Adjacent to this domain, there are regions of similarity to several RNA helicases, and to replication proteins. NS5 contains the motif Gly-Asp- Asp, which is often found in several RNA-dependent RNA polymerases. The question of whether the NS3 RNA helicase plays a role in initiation of viral replication by melting the highly conserved and stable secondary structure found at the 3'-end of DEN-2 RNA will be addressed. In vitro transcripts containing the same sequence and polarity of DEN-2 RNA, as well as model double-stranded RNA substrates will be used to characterize the NS3 helicase, followed by site-directed mutagenesis of the putative helicase domains. The NS5 RNA polymerase could potentially play a role in the viral negative strand synthesis. Model RNA substrates as template/ primers will be used to characterize the synthetic activity of NS5. Site-directed mutagenesis of the Gly-Asp-Asp domain of NS5 will be carried out to examine its functional role in RNA synthesis. All the nonstructural proteins of dengue virus have already been expressed using the recombinant vaccinia virus system. Similarly, all the structural proteins will be expressed. To dissect the protein and the nucleotide sequence requirements for initiation and elongation steps in replication, defective-interfering (DI) constructs containing the 5'-and 3'-noncoding RNA genome will be used in the presence of structural and nonstructural proteins. Understanding the mechanism of processing of the polyprotein precursors, which generates mature proteins such as NS3 and NS5, is critical to shed light on the protein requirements for genome replication. Evidence from our laboratory suggests that NS2B is required for NS3 to function as a protease in the processing of NS3->NS5 precursor involving both cis and trans cleavages. NS2B and NS3 will be purified to homogeneity using conventional as well as affinity column chromatographic methods. Several substrate analogues containing dibasic residues, as well as protease inhibitors will be used to characterize the purified NS2B/NS3 protease.