Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression
- University of Cambridge, United Kingdom
- University of Pittsburgh, United States
- Yangzhou Univeristy, China
- Imperial College London, United Kingdom
- Cambivac Ltd, United Kingdom
- University of Illinois at Urbana-Champaign, United States
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.
Data availability
Sequencing data are available on ArrayExpress under accession numbers E-MTAB-10621, E-MTAB-10622 and E-MTAB-10623. Code for the core pipeline and differential gene expression analyses was based on the pipeline available on Github at https://github.com/firth-lab/RiboSeq-Analysis.For Figure 3 (panels D-F), the raw western blots and quantification values are provided in Figure 3-source data 1-5.For Figures 4 and 5, the source data can be found in Figure 4-source data 1-3 and Figure 5-source data 1-2. The data in these tables was also used to generate Figures 7 and 8 and Figure8-figure supplements 1 and 3.For Figure 6, the source data used to annotate the ORFs is provided in Figure 6-source data 1. The data in this table was similarly used to annotate the ORFs in Figure 6-figure supplements 1 and 2 and Figure 7. The expression data in this table was used to generate some of the plots in Figure 8 and Figure 8-figure supplements 2 and 3.For Figure 11, the source data can be found in Figure 11-source data 1-3.
Article and author information
Author details
Charlotte Tumescheit
Funding
Wellcome Trust (203864/Z/16/Z)
- Georgia M Cook
Wellcome Trust (102163/Z/13/Z)
- Lior Soday
Wellcome Trust (106207/Z/14/Z)
- Andrew E Firth
Wellcome Trust (202797/Z/16/Z)
- Ian Brierley
H2020 European Research Council (646891)
- Andrew E Firth
National Institute of Food and Agriculture (2015-67015-22969)
- Ying Fang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Cook et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,839
- views
-
- 335
- downloads
-
- 16
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression
eLife 11:e75668.
https://doi.org/10.7554/eLife.75668
Further reading
-
- Epidemiology and Global Health
- Medicine
- Microbiology and Infectious Disease
eLife has published the following articles on SARS-CoV-2 and COVID-19.
-
- Cell Biology
- Chromosomes and Gene Expression
During oncogene-induced senescence there are striking changes in the organisation of heterochromatin in the nucleus. This is accompanied by activation of a pro-inflammatory gene expression programme - the senescence associated secretory phenotype (SASP) - driven by transcription factors such as NF-κB. The relationship between heterochromatin re-organisation and the SASP has been unclear. Here we show that TPR, a protein of the nuclear pore complex basket required for heterochromatin re-organisation during senescence, is also required for the very early activation of NF-κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of innate immune signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. We show that HMGA1 is also required for cytoplasmic chromatin fragment formation. Together these data suggest that re-organisation of heterochromatin is involved in altered structural integrity of the nuclear periphery during senescence, and that this can lead to activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP.
