bioRxiv preprint doi: https://doi.org/10.1101/2020.08.11.245035; this version posted February 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch Chris H. Hill*†1,3, Sawsan Napthine†1, Lukas Pekarek†2, Anuja Kibe2, Andrew E. Firth1, Stephen C. Graham*1, Neva Caliskan*2,4 and Ian Brierley*1‡ † authors contributed equally to this work * corresponding authors ‡ Lead contact:
[email protected] 1 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, UK. CB2 1QP 2 Helmholtz Institute for RNA-based Infection Research (HIRI), Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany 3 MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Ave, Cambridge, UK. CB2 0QH 4 Medical Faculty, Julius-Maximilians University Würzburg, 97074, Würzburg, Germany Abstract Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein: a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to and stabilises the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we define the conformational repertoire of this element and measure changes in unfolding pathways arising from mutation and 2A binding. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes.