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

Flaviviridae viruses are human pathogens that generate subgenomic noncoding RNAs during infection using structured exoribonuclease resistant RNAs (xrRNAs) that block progression of host cell’s exoribonucleases. The structures of several xrRNAs from mosquito-borne and insect-specific flaviviruses have been solved, revealing a conserved fold in which a ring-like motif encircles the 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. To address this, we identified subgenomic flaviviral RNA formation in the encephalitis-causing tick-borne Powassan Virus. We characterized their secondary structure using chemical probing and solved the structure of one of its xrRNAs using cryo-EM. This structure reveals a novel double loop ring element leading to a model in which the ring is remodeled upon encountering the exoribonuclease. Using bioinformatic analyses we showed that this structure is representative of a broad class of xrRNAs and defined key structural and sequence determinants of function. These discoveries reveal a conserved strategy of structure-based exoribonuclease resistance achieved through a unique topology across a viral family of key importance to global health.