The 2A protein of Theiler’s murine encephalomyelitis virus (TMEV) is required for stimulating programmed −1 ribosomal frameshifting (PRF) during infection. However, the amino acid sequence of TMEV 2A shares only 27% identity with the 2A orthologue from the related cardiovirus encephalomyocarditis virus (EMCV) for which a structure has been recently determined. Here we present the X-ray crystal structure of TMEV 2A, revealing that, despite the low sequence identity, the overall beta-shell architecture is retained, and the location of previously described mutations on this structure suggests a common RNA binding mode. We determine the minimal stimulatory element in the viral RNA required for 2A binding and show that 2A binds to this element with 1:1 stoichiometry and nanomolar affinity. We also demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence that an alternative pseudoknot-like conformation recently demonstrated for EMCV is a conserved feature of cardiovirus stimulatory elements. We go on to examine frameshifting in infected cells by ribosome profiling and metabolic labelling. We observe PRF efficiencies of up to 85%, highlighting this as the most efficient example of −1 PRF in any natural system thus far characterised. Furthermore, we document a series of ribosomal pauses in and around the site of PRF with potential implications for our understanding of translational control in cardioviruses.
Structural and molecular basis for protein-stimulated ribosomal frameshifting in Theiler’s murine encephalomyelitis virus