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.

Following infection, the (+)-stranded DENV RNA must serve as a template for replication, an mRNA for translation and a substrate for encapsidation. The function(s) assumed by these RNAs are dictated in part by the specific local and long-range RNA interactions investigated in the present study. Our combined SHAPE, aiSHAPE and Pb 2+ -induced cleavage analysis of the DENV-MINI RNA establish the existence of multiple motifs required for initiation of (À) strand RNA synthesis ( Figure 1B Figure S1 ). Our results have experimentally verified long-range interactions between the 5 0 and 3 0 terminal regions, which involved hybridization between (i) 5 0 -3 0 CS, previously demonstrated to initiate the 5 0 -3 0 long range RNA-RNA hybridization (5, 15) , (ii) the 5 0 -3 0 UAR, required for the correct orientation of the SLA with respect to the 3 0 end (30) and (iii) 5 0 -3 0 DAR, extending the CS-initiated 5 0 -3 0 interaction (11). We have predicted additional regulatory motifs that might contribute to the overall functionality of DENV-MINI RNA. Probing analysis identified tandem DBs spaced by A-rich singlestranded regions in the 3 0 -UTR, as well as internal loops and hairpins with embedded GNRA/GNRA-like motifs within the VR domain ( Figure 1B and Supplementary Figure S1 ). These elements may represent candidate assembly sites for proteins, as previous studies identified several host factors that not only bind to DENV UTRs but also actively participate in regulation of replication and translation efficiency (41) (42) (43) (44) . In addition, -GNRAtetraloops could act as 'anchors' by interacting in a sequence-specific manner with motifs at distant locations along the molecule to enhance RNA self-folding (45, 46) .