Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W.; Shapiro, Bruce A.; Padmanabhan, Radhakrishnan; Le Grice, Stuart F. J.

Current knowledge regarding the secondary structure of DENV 3 0 -UTR derives from folding algorithms and phylogenetic comparisons (8, 13) . With respect to the structure of 5 0 terminal motifs, the majority of data originate from chemical or enzymatic probing of short RNA segments (12) , often in the absence of their complementary partners (17) . In this study, we have analyzed the interplay of these regions by solving the secondary structure of DENV 5 0 and 3 0 terminal regions in the context of a 719-nt subgenomic minigenome (hereafter referred as 'DENV-MINI') (18) . We combined selective 2 0 -hydroxyl acylation analyzed by primer extension (SHAPE) with Pb 2+ ion-induced hydrolysis and site-directed mutagenesis of DENV-MINI mutant RNAs to examine the previously proposed cis-acting RNA motifs and their potential binding partners, as well as identify long-range tertiary interactions between them. Our findings provide new insights into the secondary and tertiary interactions of DENV-MINI RNA and contribute to the understanding of viral RNA replication.