Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, the viral translatome was characterised using ribosome profiling (RiboSeq), a high-throughput sequencing-based technique that allows the positions of translating ribosomes to be mapped to a genome with sub-codon precision. This was used in parallel with RNA sequencing (RNASeq) to analyse changes in viral gene expression over a timecourse of infection in MARC-145 cells.

The PRRSV genome contains two programmed ribosomal frameshift (PRF) signals, at which conserved elements promote the backwards slippage of the ribosome by one or two nucleotides. PRRSV encodes a canonical −1 PRF site, at which frameshifting is directed by a downstream RNA pseudoknot structure, to facilitate expression of the ORF1b-encoded viral replicase. At a second PRF site, both −1 and −2 PRF are stimulated by a trans-acting complex of a viral (nsp1β) and cellular (PCBP) protein in a unique, non-canonical mechanism, generating alternative forms (nsp2N and nsp2TF) of a viral non-structural protein, nsp2. Frameshift efficiency at both sites was quantified and, at the non-canonical site, was found to increase with time, rendering this the second known example of temporally regulated PRF during infection. This novel aspect of viral gene expression regulation is likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β, during the virus life cycle. Surprisingly, frameshift efficiency at the canonical ORF1ab PRF site was also found to increase over time, overturning the common assumption that RNA structure-directed sites operate at a fixed efficiency, with potential implications for the numerous other viruses which encode canonical PRF sites.

Several highly translated additional ORFs were discovered in the PRRSV genome, the translation of which is potentially facilitated by multiple novel viral transcripts. For example, a 125-codon ORF overlapping nsp12 was discovered, which is expressed as highly as nsp12 itself in the late stages of replication and likely translated from novel subgenomic (sg) RNA transcripts overlapping the 3′ end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, a highly translated, short upstream ORF (uORF) was identified in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates.

This work is the first application of RiboSeq to arterivirus-infected cells and reveals new features which add to our understanding of the complexity of gene expression programmes in this important family of nidoviruses.