De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase

Replication of positive strand flaviviruses is mediated by the viral RNA-de pendent RNA polymerases (RdRP). To study replication of dengue virus (DEN), a flavivirus family member, an in vitro RdRP assay was established using c ytoplasmic extracts of DEN-infected mosquito cells and viral subgenomic RNA templates containing 5'-and 3'-terminal regions (TRs). Evidence supported that an interaction between the TRs containing conserved stem-loop, cycliza tion motifs, and pseudoknot structural elements is required for RNA synthes is. Two RNA products, a template size and a hairpin, twice that of the temp late, were formed. To isolate the function of the viral RdRP (NS5) from tha t of other host or viral factors present in the cytoplasmic extracts, the N S5 protein was expressed and purified from Escherichia coli. In this study, we show that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo in itiation. Furthermore, the incubation temperature during initiation, but no t elongation phase of RNA synthesis modulates the relative amounts of the h airpin and de novo RNA products. A model is proposed that a specific confor mation of the viral polymerase and/or structure at the 3' end of the templa te RNA is required for de novo initiation.