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, we use 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 quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), −2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF-stimulatory viral protein (nsp1β) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in apparent 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 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) 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 temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

Women in the European Virus Bioinformatics Center

“…Her work involves screening high throughput sequencing data to identify RNA viruses, with a current focus on viruses of bees and associated arthropods. She has also worked on a number of projects on non-canonical translation in viruses [ 35 , 36 ]. She also develops bioinformatics tools, such as [ 37 ], a multiple sequence alignment cleaning and visualization tool which was co-developed with Charlotte Tumescheit (see Section 3.4 ).…”

“…cleans, interprets, and visualises multiple sequence alignments. She also performed bioinformatics analyses related to virus discovery, curation of multiple sequence alignment of viruses, and protein foldability [ 35 , 155 , 156 , 157 ].…”

Abracadabra, One Becomes Two: The Importance of Context in Viral −1 Programmed Ribosomal Frameshifting

“…The protecting ribosomes themselves are then removed and the resulting ribosome protected fragments are recovered and subjected to deep sequencing ( 34 ). As the work of Michel et al, Napthine et al and Cook et al demonstrate, these data can then be mapped onto the target gene/genome allowing one to identify locations where the ribosome may pause for extended periods (increasing numbers of reads), regions where the ribosome is less likely to be found (decreasing number of reads), and even which reading frame the ribosome is likely in (phasing) ( 35 – 37 ). Taken together this information allows the trained eye to easily identify potential PRF positions and, on the whole, is the most accurate method for determining the rate of frameshifting.…”

“…Although BYDV MFE constructs are largely nonfunctional, they still possess the expected slippery sequence-spacer-stem-loop/pseudoknot architecture that Porcine reproductive and respiratory syndrome virus (PRRSV) appears to lack ( 35 ). Indeed, beyond the requisite slippery sequence the only indication that PRRSV may undertake PRF comes from computational analyses showing significant amounts of synonymous site conservation 3′ to the slippery sequence and a striking absence of stop codons in the +1-reading frame: hallmarks of overlapping ORFs ( 54 , 55 ).…”

A frameshift in time

“…It was originally assumed that the ratio of products generated from ORF1A and ORF1B was fixed throughout the virus’s time in the cell ( Jacks and Varmus, 1985 ). Now, in eLife, Ian Brierley, Andrew Firth, Ying Fang and colleagues – including Georgia Cook (University of Cambridge) as first author – report evidence suggesting that this ratio changes over the course of infection ( Cook et al, 2022 ).…”