Abstract
Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.
Highlights
In programmed −1 ribosomal frameshifting (−1 programmed ribosomal frameshifting (PRF)), mRNA signals induce a proportion of translating ribosomes to slip back by 1 nucleotide into an overlapping open reading frame (ORF) and to continue translation, allowing the coordinated expression of two or more proteins from a single mRNA [1,2,3]
Trans-activation of porcine reproductive and respiratory syndrome virus (PRRSV) −2/−1 PRF by nsp1 was previously demonstrated by co-expression of nsp1 and nsp2 in cultured cells and by site-directed mutagenesis of the viral genome [18,19]
In rabbit reticulocyte lysate (RRL) in vitro translations, mRNAs transcribed from pDluc/PRRSV specified the synthesis of only the product of the 5 cistron of the reporter mRNA (Rluc) and no frameshifting was evident (Figure 1C)
Summary
In programmed −1 ribosomal frameshifting (−1 PRF), mRNA signals induce a proportion of translating ribosomes to slip back by 1 nucleotide (nt) into an overlapping open reading frame (ORF) and to continue translation, allowing the coordinated expression of two or more proteins from a single mRNA [1,2,3]. Central to almost all examples of −1 PRF is the interaction of the ribosome with a stimulatory mRNA structure (a stem-loop or RNA pseudoknot) which promotes frameshifting on a stretch of homopolymeric bases known as the slippery sequence. How these RNA structures act is incompletely understood, but accumulating evidence supports the view that by presenting an unusual topology [1,2,8,9,10,11] they confound an intrinsic unwinding activity of the ribosome with consequent effects on the elongation cycle and frame maintenance [12,13,14]. Kinetic analyses in bacterial systems indicate that stimulatory RNAs can impair movements of the ribosomal small subunit (30S) head, delaying dissociation of EF-G and the release of tRNA from the ribosome [15,16,17]
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