Abstract
Many viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.
Highlights
Programmed ribosomal frameshifting (PRF) is a gene expression mechanism whereby a proportion of translating ribosomes are stimulated to shift into an alternative reading frame at a specific site during the decoding of an mRNA [1]
To test whether 2A stimulation was specific to the cardiovirus PRF signal, reporter mRNAs containing the human immunodeficiency virus 1 (HIV; family Retroviridae) or infectious bronchitis virus (IBV; family Coronaviridae) frameshift signals [28] were translated in wheat germ (WG) or rabbit reticulocyte lysate (RRL) with or without recombinant Theiler’s murine encephalomyelitis virus (TMEV) 2A protein
Like encephalomyocarditis virus (EMCV), −1 PRF in TMEV is transactivated by the viral 2A protein
Summary
Programmed ribosomal frameshifting (PRF) is a gene expression mechanism whereby a proportion of translating ribosomes are stimulated to shift into an alternative reading frame at a specific site during the decoding of an mRNA [1]. Ribosomes which frameshift produce an alternative protein product that is N-terminally coincident with the product of standard decoding but has a distinct C-terminal region encoded by either the +1 or the −1 reading frame depending on the type of frameshifting. The most common type of PRF involves −1 nt tandem slippage of the P- and A-site tRNAs on the mRNA (−1 PRF). Other viruses use −1 PRF to append an extension domain onto a proportion of their capsid proteins A number of cellular genes utilize −1 PRF in their expression, both in eukaryotes [6,7] and in bacteria [8,9]
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