MOTIVATION: The Flavivirus genus includes several important pathogens, such as Zika, dengue and yellow fever virus. Flavivirus RNA genomes contain a number of functionally important structures in their 3' untranslated regions (3'UTRs). Due to the diversity of sequences and topologies of these structures, their identification is often difficult. In contrast, predictions of such structures are important for understanding of flavivirus replication cycles and development of antiviral strategies. RESULTS: We have developed an algorithm for structured pattern search in RNA sequences, including secondary structures, pseudoknots and triple base interactions. Using the data on known conserved flavivirus 3'UTR structures, we constructed structural descriptors which covered the diversity of patterns in these motifs. The descriptors and the search algorithm were used for the construction of a database of flavivirus 3'UTR structures. Validating this approach, we identified a number of domains matching a general pattern of exoribonuclease Xrn1-resistant RNAs in the growing group of insect-specific flaviviruses. AVAILABILITY AND IMPLEMENTATION: The Leiden Flavivirus RNA Structure Database is available at https://rna.liacs.nl. The search algorithm is available at https://github.com/LeidenRNA/SRHS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The intergenic regions of the ambisense RNA segments of viruses from the Tospovirus genus form large extended RNA structures that regulate virus replication. Using comparative structure analysis, we show the presence of conserved alternative conformations at the apical parts of these structures. In one conformation, a branched Y-shape, the 5'-proximal hairpin arms are mostly capped by exceptionally stable tetraloop motifs. The tetraloop hairpins are folded in both virus and virus-complementary sense RNAs, and different tetraloops can functionally replace each other. Folding simulations show that the branched Y-shape structures can undergo a conformational transition to alternative extended rod-like conformations. Functional importance of both alternatives is supported by nucleotide covariations. The balanced equilibrium between alternative structures is evidenced by native gel electrophoresis of mutant RNA transcripts with shifted equilibria. The tetraloops play a role in the stability and dynamics of structures but may also be recognized by proteins involved in translation and/or replication.

UNLABELLED: Recent outbreaks of avian influenza are being caused by unusually virulent H5N1 strains. It is unknown what makes these recent H5N1 strains more aggressive than previously circulating strains. Here, we have compared more than 3000 RNA sequences of segment 8 of type A influenza viruses and found a unique single nucleotide substitution typically associated with recent H5N1 strains. By phylogenetic analysis, biochemical and biophysical experiments, we demonstrate that this substitution dramatically affects the equilibrium between a hairpin and a pseudoknot conformation near the 3' splice-site of the NS gene. This conformational shift may have consequences for splicing regulation of segment 8 mRNA. Our data suggest that besides changes at the protein level, changes in RNA secondary structure should be seriously considered when attempting to explain influenza virus evolution. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.