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.

Minimal free-energy modeling using SHAPE data as pseudo free-energy constraints is consistent with proposed 5 0 -UTR motifs in the context of DENV-MINI RNA ( Figure 1B and Supplementary Figure S1 ) (6, 17, 30) . Two regions that exhibit high sensitivity to NMIA modifications were located within the 5 0 stem-loop A (SLA), A31-U34 and U48-A50, which corresponded to the TL and the side stem-loop (SSL), respectively. Three helical regions of 5 0 SLA, U3-G9:C64-G70, C11-G15:A56-G61 and C24-A29:U35-G40, were insensitive to NMIA. The remainder of the reactive nucleotides were situated within the U62-U63 bulge and internal loops G17-A19 and G22-A23. Downstream of SLA, the oligoU tract spanning nt U71-U80 was predicted to be singlestranded. This region was followed by the unreactive double-stranded upstream of AUG region (dsUAR), formed via a long-range interaction between the 5 0 -and 3 0 -UTRs (nt A81-G96 and nt C638-U654). Here, the only reactive residues occupied the vicinity of bulge (C646), mismatch (G84) and the closing base pair of the helix. The internal loop enclosing the first AUG codon (nt A97-A104 and G631-C637) was predicted to be singlestranded followed by the double-stranded 6-nt long downstream of UAR region (DAR) containing reactive residues within the mismatch (A108) and the closing pairs of the helices. The capsid hairpin (cHP, A115-U133) (12), displayed an unreactive stem followed by a 7-nt reactive apical loop. As suggested by previous chemical probing studies, the 5 0 -3 0 terminal interactions were mediated by base pairing between the 5 0 and 3 0 cyclization sequences, which formed 11-nt double-stranded cyclization region (CS) (U134-G144 and C614-A624) (12) . Subsequently, two short hairpins that formed downstream of the 5 0 -3 0 CS were found within the sequence of the 5 0 -end of the capsid protein gene. One contains a purine-rich and moderately reactive apical loop (A152-U159) and the other with a reactive 3-nt loop and A-C mismatch (G165-C185). Both hairpins were followed by 5-nt helical region (A190-U194 and A444-U448), which provided additional linkage between the 5 0 and 3 0 termini.