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.

Pb 2+ ion-induced cleavage pattern for mutant PK1 Flip substantiated the SHAPE-predicted alterations of RNA secondary structure ( Figure 5 insert and Supplementary Figure S9 ). Residues of SLB apical loop (A85-A88), as well as internal loop (U73-A78 and U99-U100), were readily hydrolyzed, whereas the A78-G84:U91-U98 stem was not cleaved. Also, the DAR region (A107-C109) and 5 0 CS (A137-C142) were susceptible to cleavage, which agreed with their single-stranded character. With regard to the 3 0 -UTR, moderate hydrolysis was noted within the stem of the extended 3 0 SL, as it contained numerous bulges and loops that relaxed its overall stability. Pb 2+ cleavage was most pronounced within the apex of the 3 0 SL (A674-C675). The 3 0 CS region containing the flipped PK1 exhibited complementarity between TL2 and PK1. Also, SHAPEassisted prediction of RNA folding showed that PK1 was embedded in single-stranded region available for pairing with TL2. It was suggested previously that releasing PK1 from the 5 0 -3 0 CS region provides the switch between the translation and replication mode of the DENV genome by rendering PK1 available for a pseudoknot interaction with TL2 (13) . In our study, the RNA secondary structure predicted for TL2 Flip PK1 Flip mutant resembled the structure of the PK1 Flip mutant ( Supplementary Figures S10 and S11) . Residues of neither PK1 nor TL2 displayed a change in NMIA sensitivity, contradicting the implication that TL2 is involved in long-range interactions with PK1 in the context of DENV-MINI construct. Pb 2+ -induced hydrolysis of TL2 Flip PK1 Flip mutant provided a pattern of cleavage comparable with that obtained for the PK1 Flip mutant (Supplementary Figure S12) , the only difference being lower intensity of hydrolysis within the internal loops and rightmost apical loops of both DBs.