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.

The region upstream of the DBs is often referred to as the variable region (VR), as its sequence differs between DENV serotypes ( Figure 1B and Supplementary Figure S1 ) (33) . Our probing analysis indicated its propensity to form long single-stranded stretches (A274-A291, U364-A376, G418-C424 and U439-A443). One of the loops (U439-A443), composed almost exclusively of adenine residues, was mainly NMIA unreactive. It was shown previously that consecutive adenine bases in single-stranded regions are often involved in stacking interactions, which would account for their inaccessibility towards NMIA modification (34, 35) . The 3 0 -UTR region also contained two stable tetraloops (C352-G363 and C425-G438). As expected, the -GNRA-tetraloop (C425-G438), restricted by extensive stacking and hydrogen-bonding interactions, was NMIA-insensitive (31) . On the other hand, the -CAAG-tetraloop (C352-G363) behaved differently. Here, only its GC residues showed little reactivity, which could be interpreted as formation of an intraloop G · C base pair, whereas its AA residues were reactive. The apical loops of two other hairpins (G377-C392 and G396-C412) showed strong NMIA reactivity. Also, an extended hairpin containing internal asymmetric loop (A296-U301 and U345-G346), a bulge (C307-A313) and A-rich, moderately reactive apical loop (A323-A331) was predicted to form between nucleotides G292-C350. Another long hairpin (U206-A267) with a reactive apical loop (A233-A241) and two reactive internal loops (G218-A222 and C252-A256; A227-A229 and G245-A247) enclosed a linker sequence that stretched from A227-G268.