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 a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. 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. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
Highlights
Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that inhibits cap-dependent translational initiation
Through site-directed mutagenesis and the use of single-molecule optical tweezers, we study the dynamics of this RNA element, both alone and in the presence of 2A
By observing short-lived intermediate states in real-time, we demonstrate that the encephalomyocarditis virus (EMCV) stimulatory element exists in at least two conformations and 2A-binding stabilises one of these, an RNA pseudoknot, increasing the force required to unwind it
Summary
Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that inhibits cap-dependent translational initiation. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection. The spacing between the slippery sequence and stem-loop is 13 nt, significantly longer than typically seen, and 2A protein has been proposed to bridge this gap through interaction with the stem-loop[12] This allows for temporal control of gene expression as the efficiency of –1 frameshifting is linked to 2A concentration, which increases with time throughout the infection cycle[12]. We show that 2A binds directly to the frameshift-stimulatory element in the viral RNA with nanomolar affinity and equimolar stoichiometry, and we define the minimal
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