Ribosome profiling of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of huge economic importance, infecting the porcine host leading to infertility, morbidity, and mortality. Its genome contains a canonical −1 programmed ribosomal frameshift (PRF) site that facilitates expression of the viral replicase, and a second, non-canonical signal which induces both −1 and −2 PRF to generate alternative forms of a viral non-structural polypeptide, nsp2. In contrast to canonical frameshift sites, the frameshift at the non-canonical site is not stimulated by a downstream secondary RNA structure, but is instead the first known example of protein-directed frameshifting, stimulated by a trans-acting complex of a viral (nsp1β) and a cellular (PCBP) protein. We investigated frameshifting in PRRSV by ribosome profiling, using the vaccine strain, SD95-21, and a derivative with mutations at the nsp2 site that render it PRF-defective. Highly efficient PRF was observed at both the canonical, RNA pseudoknot-dependent −1 PRF site (efficiency of43–56 %), and the nsp1β/PCBP-dependent site (combined −1 and −2 PRF efficiency of 20–24 %). Investigations are underway as to whether the presence of nsp1βduring viral infection stimulates non-canonical frameshifting on host mRNAs. We also carried out RNA-Seq and differential transcription analysis in parallel with ribosome profiling to garner further insight into the functions of thensp2transframe proteins, previously shown to be involved in innate immune suppression. This ribosome profiling analysis has also revealed the presence of a short but highly expressed upstream ORF in the 5’UTR of PRRSV.