PT  - JOURNAL ARTICLE
AU  - Chris H. Hill
AU  - Georgia M. Cook
AU  - Sawsan Napthine
AU  - Anuja Kibe
AU  - Katherine Brown
AU  - Neva Caliskan
AU  - Andrew E. Firth
AU  - Stephen C. Graham
AU  - Ian Brierley
TI  - Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in <em>Theilovirus</em>
AID  - 10.1101/2020.08.11.245068
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2020.08.11.245068
4099  - http://biorxiv.org/content/early/2021/03/18/2020.08.11.245068.short
4100  - http://biorxiv.org/content/early/2021/03/18/2020.08.11.245068.full
AB  - The 2A protein of Theiler’s murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed −1 ribosomal frameshifting (PRF) during infection. Here we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinning Theilovirus protein-stimulated frameshifting.Competing Interest StatementThe authors have declared no competing interest.