Introduction. Accurate maintenance of the translational reading frame by ribosomes is essential for the production of functional proteins and unsurprisingly, errors in frame maintenance have been estimated to occur at rates probably lower than 5 × 10-5 per codon (Kurland, 1992). However, an increasing number of examples have been documented where a purposeful shift in reading frame is programmed into the mRNA and serves an essential function (Gesteland et al., 1992; Atkins & Gesteland, 1995). Highly efficient ribosomal frameshifting is an example of such a programmed frameshift site. In response to certain signals in the mRNA, ribosomes are induced to move into the -1 reading frame (in a 5′ direction) at a specific point and continue translation in the new reading frame. The phenomenon was first described in 1985 as the way in which the Gag-Pol polyprotein of the retrovirus Rous sarcoma virus (RSV) is expressed from the overlapping gag and pol open reading frames (ORFs) (Jacks & Varmus, 1985) and to date, most examples of frameshifting come from virus systems.

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. Ribosome profiling is emerging as a powerful technique to monitor translation in living cells at sub-codon resolution. It has particular applicability to virology, with the capacity to identify viral mRNAs that are being translated during infection and to provide new insights into virus gene expression, regulation and host-virus interactions. In this work, we carried out the first ribosome profiling analysis of an RNA virus, using as a model system the murine coronavirus strain MHV-A59, a betacoronavirus in the same genus as the medically important SARS-CoV and MERS-CoV. Parallel ribosome profiling and RNA sequencing of infected-cell time points was performed during the course of MHV replication in mouse tissue culture cells and used to determine virus gene expression kinetics and the relative translational efficiencies of virus and host mRNAs. The sensitivity and precision of the approach permitted us to uncover several unanticipated features of coronavirus translation, giving insights into ribosomal frameshifting, ribosome pausing, and the utilisation of short, potentially regulatory, upstream open reading frames. We also identified some challenges associated with the technique that are of general relevance to the ribosome profiling technique and developed bioinformatic strategies to address these.

Significance: Ribosomes synthesize proteins by translating mRNAs into linear chains of amino acids through the decoding of consecutive nucleotide triplets (codons). Specific mRNA signals, however, can stimulate ribosomes to shift into an alternative triplet reading frame (ribosomal frameshifting) resulting in translation of a different protein. Typically, such signals are regions of intramolecular nucleotide base-pairing in the mRNA which form structures that stall ribosome progress. Here we show that the frameshifting signal used to express the nsp2TF and nsp2N proteins of porcine reproductive and respiratory syndrome virus, an important swine pathogen, requires the action of a transacting viral protein rather than a structured RNA. This novel mechanism of gene expression may also be used by other viruses or in cellular gene expression.

Here we investigated ribosomal pausing at sites of programmed -1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.