Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

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.

• Authors: Chris H Hill, Georgia M Cook, Sawsan Napthine, Anuja Kibe, Katherine Brown, Neva Caliskan, Andrew E Firth, Stephen C Graham, Ian Brierley
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 49 Issue 20 Pg. 11938-11958 (11 18 2021) ISSN: 1362-4962 [Electronic] England
• PMID: 34751406 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chemical References

• Viral Proteins
• virus protein 2A

Topics

• Frameshifting, Ribosomal
• Ribosomes (metabolism)
• Theilovirus (genetics, metabolism)
• Viral Proteins (chemistry, metabolism)