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.

SHAPE-directed prediction of RNA folding indicated similar structure rearrangements for the TL2 Flip PK1 Flip mutant RNA (Supplementary Figures S10-S12) , suggesting PK1 is mostly responsible for correct long-range communication between the 5 0 and 3 0 terminal regions. Even the presence of compensatory mutations within 5 0 CS (TL2 Flip PK1 Flip -3 mutant) to restore base pairing with PK1 ( Figure 6 and Supplementary Figures S13-S15) did not entirely reinstate the secondary structure of DENV-MINI RNA. Previous studies using DENV-MINI RNA as templates in in vitro RdRp assays have shown that replication of PK1 Flip , TL2 Flip PK1 Flip mutants was significantly affected, supporting the importance of the 5 0 -3 0 CS interaction for RNA synthesis (8) . However, the introduction of compensatory mutations at the 5 0 CS in DENV-MINI TL2 Flip PK1 Flip -3 mutant restored replication to nearly wild-type level. This was in contrast to the effects of the PK1 'flipping' mutations on replication of luciferase-based DENV2 in BHK-21 cells, which showed essentially no replication (8) . Related studies using luciferase containing Dengue virus (15) , Kunjin virus (57) and West Nile virus replicons (58) carrying point mutations at the 3 0 CS are consistent with our observations. In all cases, the altered sequence of 3 0 CS attenuated replication, even when mutations were compensated at the 5 0 CS. Also, a mutation introduced into the 3 0 CS extending the region of complementarity between 5 0 -3 0 CS by one nucleotide was shown to negatively affect replication kinetics of the subgenomic DENV reporter replicon (59) . Apparently, not only the secondary structure of 5 0 -3 0 CS region but also its sequence composition governs the efficiency of RNA synthesis.