Tospovirus ambisense genomic RNA segments use almost complete repertoire of stable tetraloops in the intergenic region.

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.