Tick-borne flavivirus exoribonuclease-resistant RNAs contain a double loop structure

Viruses from the Flaviviridae family contain human relevant pathogens that generate subgenomic noncoding RNAs during infection using structured exoribonuclease resistant RNAs (xrRNAs). These xrRNAs block progression of host cell's 5' to 3' exoribonucleases. The structures of several xrRNAs from mosquito-borne and insect-specific flaviviruses reveal a conserved fold in which a ring-like motif encircles the 5' end of the xrRNA. However, the xrRNAs found in tick-borne and no known vector flaviviruses have distinct characteristics, and their 3-D fold was unsolved. Here, we verify the presence of xrRNAs in the encephalitis-causing tick-borne Powassan Virus. We characterize their secondary structure and obtain a mid-resolution map of one of these xrRNAs using cryo-EM, revealing a unique double-loop ring element. Integrating these results with covariation analysis, biochemical data, and existing high-resolution structural information yields a model in which the core of the fold matches the previously solved xrRNA fold, but the expanded double loop ring is remodeled upon encountering the exoribonuclease. These results are representative of a broad class of xrRNAs and reveal a conserved strategy of structure-based exoribonuclease resistance achieved through a unique topology across a viral family of importance to global health.