In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5 '- and 3 '-terminal regions that influence RNA structure

Viral replicases of many positive-strand RNA viruses are membrane-bound com plexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our res ults indicated that in vitro RNA synthesis at the 5'-untranslated region (U TR) required the presence of the 5'-terminal region (TR) and the two cycliz ation (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV crosslinking method and an in vitro RdRP as say, we analyzed structural determinants for physical and functional intera ctions. Exogenous RNA templates that were used in the assays contained dele tion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot struc ture. Our results indicate that there is physical interaction between the 5 '-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'- UTR, however, requires long range interactions involving the 5'-UTR, CYC mo tifs, and the 3'-stem-loop region that includes the tertiary pseudoknot str ucture.