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Abstract
Programmed −1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3′-adjacent to a ‘slippery’ heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of −1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3′-adjacent stem-loop structure, but is not appreciably affected by the ‘StopGo’ sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46–76 % efficient.
Highlights
Programmed À1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift À1 nt and continue translating in the new reading frame
Like other members of the family Picornaviridae, encephalomyocarditis virus (EMCV) has a single-stranded RNA genome of positive polarity which serves as an mRNA
Translation initiation is mediated by an internal ribosome entry site (IRES) within the 5¢ UTR
Summary
Programmed À1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift À1 nt and continue translating in the new reading frame. BHK-21 or L929 cells were infected with V5-tagged WT or mutant viruses at m.o.i.~10 and lysates were prepared at 8 h p.i. Mu-anti-2A detected a product migrating just above 15 kDa for V5-WT and V5-SS viruses (Fig. 1c, right, lanes 1–2).
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