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.

With regard to the 3 0 -UTR, SHAPE provided information on the 3 0 SL structure, obscured only at the extreme 3 0 terminus because of the presence of hybridized primer ( Figure 1B and Supplementary Figure S1 ). The 3 0 SL displayed NMIA sensitivity at the apical loop (A674-C680) and insensitivity at its leftmost side. The fact that this large 79-nt stem-loop was followed with reactive stretch of 3 nt (C655-U657) and unreactive dsUAR region (nucleotides A81-G96 and nucleotides C638-U654), enforced the proposed conformation. Previous phylogenetic comparative studies suggested that the 3 0 -UTR enclosed two almost identical DBs that might assume a pseudoknot conformation with their downstream complementary regions (13, 16) . SHAPE data confirmed the presence of both, and in addition provided detailed insight into their secondary structure. The 3 0 DB contained a 6-bp unreactive stem (U536-G541 and C598-A603) and two hairpins on opposing ends (G547-U571 and G584-C597). The apical loop of the leftmost hairpin incorporated the highly reactive TL2 sequence (G558-U562). The reactivity of TL2, combined with the fact that its downstream complementary partner PK1 (A613-C617) was involved in formation of 5 0 -3 0 CS region, would likely prevent the previously suggested TL2/PK1 pseudoknot interaction (13, 16) . Regarding the 5 0 DB, it likewise contained an unreactive stem (G449-G454 and C511-C516) and two hairpins on opposing ends (C462-G482 and G497-C510). Here, however, the apical loop of the leftmost hairpin incorporating the TL1 sequence (G467-G477), although predicted to be single-stranded, harbored mainly unreactive nucleotides. The fact that its complementary partner PK2 was likewise unreactive and enclosed within single-stranded loop (G526-C530) was strongly suggestive of a TL1/ PK2 base pairing interaction ( Figure 2 ) (13, 16) . In addition, both DBs comprised reactive bulges (A491-C496 and A578-A583) and moderately NMIA-sensitive purine-rich internal loops. One of the internal loops of the 3 0 DB (G572-A575) was insensitive to modification as it contained a -GGAA-motif. Such -GNRA-tetraloops represent a unique fold, which include a trans sugar edge/ Hoogsteen edge G · A pair between the first and the last residues, a hydrogen bond between the 2 0 -OH of G and the N7 of A and 3 0 stacking of all tetraloop bases except the G. This architecture would account for lack of reactivity towards NMIA (31) .