Mechanical strength of RNA knot in Zika virus protects against cellular defenses

Unusual knot-like structures recently discovered in viral exoribonuclease-resistant RNAs (xrRNAs) prevent digestion by host RNases to create subgenomic RNAs enhancing infection and pathogenicity. xrRNAs are proposed to prevent digestion through mechanical resistance to unfolding. However, their unfolding force has not been measured, and the factors determining RNase resistance are unclear. Furthermore, how these knots fold remains unknown. Unfolding a Zika virus xrRNA with optical tweezers revealed that it was the most mechanically stable RNA yet observed. The knot formed by threading the 5' end into a three-helix junction before pseudoknot interactions closed a ring around it. The pseudoknot and tertiary contacts stabilizing the threaded 5' end were both required to generate extreme force resistance, whereas removing a 5'-end contact produced a low-force knot lacking RNase resistance. These results indicate mechanical resistance plays a central functional role, with the fraction of molecules forming extremely high-force knots determining the RNase resistance level.