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.

A 3D structural model of the DENV-MINI RNA was generated using RNAComposer, a fully automated server that predicts 3D structural models from RNA secondary structure (40) (Figure 7) . As the server does not accept sequences longer than 500 nt, a 474-nt derivative sequence was created by deleting the VR domain and closing the remaining short helical region (A190-U194 and A443-U448) with a -GAGA-tetraloop. A dotbracket notation generated by the RNAstructure software was adjusted to account for this internal deletion, as well as the predicted TL1/PK2 pseudoknot, and subsequently provided to RNAComposer. Ten 3D RNA models were generated and analyzed, taking into account their secondary structure topology, sequence homology, structure resolution and free energy. In addition, the quality of predicted models was evaluated using the MolProbity tool (24) . The best of these models was manually adjusted to correct for an unlikely fold within PK1, and the affected regions were subjected to energy minimization (Accelrys Discovery Studio v3.5) to create the structure of Figure 7 .