Exoribonuclease-resistant RNAs (xrRNAs) from viruses prevent digestion by host exoribonucleases, creating sub-genomic viral RNAs that can enhance infection and pathogenicity. Novel knotted structures in xrRNAs are proposed to act as mechanical roadblocks to RNases. Studying an xrRNA from Zika virus with optical tweezers, we found that it was the most mechanically stable RNA structure yet observed. The knot folded by threading the 5′ end into the cleft of a Mg2+-coordinated three-helix junction before pseudoknot interactions closed a ring around it. Both the threading and pseudoknot were required to generate the extremely force-resistant knot, whose formation correlated directly with RNase resistance both in the wild-type xrRNA and a low-resistance mutant. This work clarifies the folding and mechanism of action of an important new class of RNA. ### Competing Interest Statement The authors have declared no competing interest.
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