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.

In addition, the TL2 Flip PK1 Flip mutant (Supplementary Figure S10) provided insight into the second, previously proposed TL2/PK1 pseudoknot (13, 16) . It was suggested previously that PK1 could modulate DENV genome cyclization via either a 5 0 -3 0 CS interaction or by base pairing with TL2 (13, 25) . Also, studies showed that the longdistance 5 0 -3 0 CS complementarity interferes with efficient translation of DENV2 luciferase (LUC) reporter system in Vero cells (25) . It was suggested that the 5 0 -3 0 CS interaction may play role in regulating the transition from the translation to the replication phase of the infection. Thus, we expected that release of PK1 from 5 0 -3 0 CS together with the conserved complementarity between both partners, would support formation of TL2/PK1 pseudoknot. SHAPE analysis, however, indicated no reactivity change within 'flipped' TL2 and PK1 (Supplementary Figures S10 and S11) . As the addition of 'flipping' mutations within TL2 did not cause any further structural rearrangements compared with PK1 Flip mutant (compare Figure 5 and Supplementary Figures S7 and S8) , this would dismiss the involvement of TL2 in tertiary interactions. However, we cannot exclude the possibility that in vivo a TL2/PK1 interaction could be stabilized by participation of host or viral factors for the purpose of other molecular processes, i.e. translation, before the 5 0 -3 0 CS interaction. Indeed, it has been proposed that the viral NS3 helicase can modulate the folding of cis-acting RNA elements via dual RNA unwinding/annealing activities, providing the switch between alternative conformations of the DENV genome (38, 60) .