Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus. Issue 20 (9th November 2021)

• Record Type: Journal Article
• Authors: Hill, Chris H; Cook, Georgia M; Napthine, Sawsan; Kibe, Anuja; Brown, Katherine; Caliskan, Neva; Firth, Andrew E; Graham, Stephen C; Brierley, Ian
• Is Part Of: Nucleic acids research. Volume 49:Issue 20(2021)
• Page Start: 11938
• Page End: 11958
• Publication Date: 2021-11-09
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkab969
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: 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.