Conserved long-range interactions are required for stable folding of orthoflaviviral genomic RNA.

Long-range tertiary interactions are a widespread structural feature in viral RNAs (vRNAs) and mRNAs. In the orthoflaviviruses, conserved complementary sequences in the 5' and 3' terminal regions have an essential role in viral replication. Long-range pairing of these conserved sequences is proposed to facilitate a switch between two alternative vRNA conformations. Yet the detailed nature of these interactions, their relative populations and their exchange are required to formulate a mechanistic model of their role in regulation of the viral life cycle. Here, we used single-molecule Förster resonance energy transfer to study the global conformation of vRNAs by measuring their end-to-end distances. We observed that vRNA conformation is heterogeneous, and that conformers with close end-to-end distances have unusual kinetic stability when compared with mRNA lacking these specific long-range interactions. vRNAs also partition between at least two stable states with a large rearrangement of the terminal regions (>50 à change in end-to-end distance). We demonstrate that this bistability depends on long-range interactions and is modulated by host factors such as the initiation factor complex eIF4F. Understanding how vRNA and its stability is influenced by interactions with other host and viral factors will help to elucidate a mechanistic role for these highly conserved orthoflaviviral sequences.