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.
Highlights
Cardioviruses are a diverse group of picornaviruses that cause encephalitis, myocarditis and enteric disease in a variety of mammalian hosts including rodents, swine and humans [1]
The Cardiovirus B or Theilovirus species comprises several isolates including Sikhote-Alin virus [2], rat theilovirus and Theiler’s murine encephalomyelitis virus (TMEV), all of which are genetically distinct from the Cardiovirus A species encephalomyocarditis virus (EMCV) [3]
We provide evidence that the alternative pseudoknot-like conformation recently described for the EMCV stimulatory element [28] is likely to be present in TMEV and other cardioviruses
Summary
Cardioviruses are a diverse group of picornaviruses that cause encephalitis, myocarditis and enteric disease in a variety of mammalian hosts including rodents, swine and humans [1]. Several ‘non-canonical’ translation events occur during the production of the TMEV polyprotein. A cotranslational StopGo or ribosome ‘skipping’ event occurs at the junction between the 2A and 2B gene products [9,10]. In this process, the peptidyl-transferase reaction fails between the glycine and proline in a conserved D(V/I)ExNPG|P motif, releasing the upstream L-1ABCD-2A product as the ribosome continues translating the downstream 2BC3ABCD region. It has been hypothesised that in TMEV, the main function of PRF may be to downregulate translation of the enzymatic proteins encoded downstream of the frameshift site, in the late stages of infection [11]
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