Dengue virus (DV) is a current global health problem affecting tropical and sub-tropical regions. The existence of four dengue serotypes renders individuals who are partially protected against one serotype to be even more vulnerable to severe disease caused by another. Therefore, development of a vaccine will require a broad mechanism for virus attenuation. Dengue virus non-structural protein 5 (DV NS5) is the viral RNA-dependent RNA polymerase (RdRp). The poliovirus system has suggested that the fidelity of a viral RdRp can be targeted for viral attenuation and vaccine development. Most studies of Dengue non-structural protein 5 (DV NS5) have focused on cis-acting elements or qualitative determinants of polymerase function. The goal of this study was to acquire the first quantitative perspective of DV NS5 structure-function relationships. The major findings were: (1) The methyltransferase domain of DV NS5 is required for optimal RdRp activity; (2) DV NS5 has a preference for triphosphorylated primers; (3) A conserved motif-D lysine is required for nucleotidyl transfer efficiency in DV NS5. In addition, an assay to characterize the kinetics of nucleotide binding and elongation has been developed.
