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

View ORCID ProfileChris H. Hill, View ORCID ProfileGeorgia M. Cook, Sawsan Napthine, Anuja Kibe, View ORCID ProfileKatherine Brown, View ORCID ProfileNeva Caliskan, View ORCID ProfileAndrew E. Firth, View ORCID ProfileStephen C. Graham, View ORCID ProfileIan Brierley
doi: https://doi.org/10.1101/2020.08.11.245068
Chris H. Hill
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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  • For correspondence: ib103@cam.ac.uk
Georgia M. Cook
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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Sawsan Napthine
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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Anuja Kibe
2Helmholtz Institute for RNA-based Infection Research (HIRI), Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany
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Katherine Brown
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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Neva Caliskan
2Helmholtz Institute for RNA-based Infection Research (HIRI), Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany
3Medical Faculty, Julius-Maximilians University Würzburg, 97074, Würzburg, Germany
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Andrew E. Firth
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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  • For correspondence: ib103@cam.ac.uk
Stephen C. Graham
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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  • For correspondence: ib103@cam.ac.uk
Ian Brierley
1Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP
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  • For correspondence: ib103@cam.ac.uk
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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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Minor improvements to text and figures to enhance clarity

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted March 18, 2021.
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Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus
Chris H. Hill, Georgia M. Cook, Sawsan Napthine, Anuja Kibe, Katherine Brown, Neva Caliskan, Andrew E. Firth, Stephen C. Graham, Ian Brierley
bioRxiv 2020.08.11.245068; doi: https://doi.org/10.1101/2020.08.11.245068
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Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus
Chris H. Hill, Georgia M. Cook, Sawsan Napthine, Anuja Kibe, Katherine Brown, Neva Caliskan, Andrew E. Firth, Stephen C. Graham, Ian Brierley
bioRxiv 2020.08.11.245068; doi: https://doi.org/10.1101/2020.08.11.245068

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