Abstract
The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-sense, single[49] stranded RNA virus in the family Arteriviridae[1,2], and the aetiological agent of the disease from which it takes its name: porcine reproductive and respiratory syndrome (PRRS)
PRRSV gene expression was investigated using three viruses: an EU PRRSV isolate based on the 136 Porcilis® vaccine strain (MSD Animal Health; GenBank accession OK635576.1), NA PRRSV SD95137 21 (GenBank accession KC469618.1), and a previously characterised mutant variant (NA PRRSV SD95-21 KO2) which bears silent mutations in the nsp[2] programmed ribosomal frameshifting (PRF) site slippery sequence and C-rich motif rendering it unable to bind poly(rC) binding protein (PCBP), induce −1 or −2 PRF, or produce nsp2N or nsp2TF (Figure 1A, inset)[23–25,32]
This work is the first application of ribosome profiling to an arterivirus, and has revealed a complex complement of PRRSV gene expression strategies, several of which permit stoichiometric modulation of the polyprotein proteins
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive-sense, single[49] stranded RNA virus in the family Arteriviridae (order: Nidovirales)[1,2], and the aetiological agent of the disease from which it takes its name: porcine reproductive and respiratory syndrome (PRRS). The two lineages of PRRSV, formerly known as “European” (Type 1) and “North American” (Type 2) PRRSV, share just ~60% pairwise nucleotide similarity and were recently re-classified as two separate species, Betaarterivirus suid 1 and 2 (viruses named PRRSV-1 and PRRSV-2)[5–7]. Despite the substantial genetic and antigenic diversity between the two species, the overall clinical symptoms are similar, there is considerable (~20%) genetic diversity within each species, rendering this isolate-dependent[3,5]. This is largely due to PRRSV’s rapid mutation rate, which leads to relatively frequent emergence of highly pathogenic strains capable of escaping existing immunity, within the NA PRRSV species[3,5]
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