Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression
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Evaluation Summary:
The paper presents the first comprehensive gene expression analysis of two species of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes economically important diseases of pigs. By applying ribosome profiling to arterivirus-infected cells the authors discovered a substantial number of efficiently translated open reading frames in viral RNAs and obtained compelling evidence of changes in ribosome frameshifting efficiencies over the course of infection. The paper is of interest to virologists and researchers studying mRNA decoding, recoding, and translation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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.
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Author Response
Reviewer #1 (Public Review):
The authors use ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) to characterise the transcriptome and translatome of two PRRSV species as well as the host in response to infection. One particularly exciting feature of the study is that the analysis is carried out at different times of infection, which shows how both the virus and the host regulate their gene expression. The authors identify several new regulatory mechanisms of virus gene expression. Unexpectedly, they also find that the frameshifting efficiency at the ORF1ab frameshifting site changes with time. This contradicts the dogma in the field, which states that frameshifting is constant and has evolved to be constant to produce the a particular ratio of the two protein isoforms. The strength of the paper is in its …
Author Response
Reviewer #1 (Public Review):
The authors use ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) to characterise the transcriptome and translatome of two PRRSV species as well as the host in response to infection. One particularly exciting feature of the study is that the analysis is carried out at different times of infection, which shows how both the virus and the host regulate their gene expression. The authors identify several new regulatory mechanisms of virus gene expression. Unexpectedly, they also find that the frameshifting efficiency at the ORF1ab frameshifting site changes with time. This contradicts the dogma in the field, which states that frameshifting is constant and has evolved to be constant to produce the a particular ratio of the two protein isoforms. The strength of the paper is in its comprehensible analysis. The paper is extremely rich in data, with 12 main and 23 Supplemental Figs and 11 Supplemental Tables, all of them rather complex. The main weakness is that it is written in a technical language that will be hardly readable by a non-specialist readership. Unfortunately, the authors do not make a good job in guiding the reader through their findings and hardly identify the the most important findings, while leaving the details to the specialists. This is particularly exemplified in Fig. 12, which should present the summary of the findings and would be extremely helpful, but hardly provides any text at all. This is potentially a very interesting paper, but the impact on the field could be increased considerably by better presentation of the work.
We would like to thank this reviewer for the positive comments about the scientific findings, and for their suggestions for improving the presentation of the work. This outside perspective was very useful in helping us see which parts of the paper required clearer explanation or less detail, which can be hard to discern when very close to the work. We have incorporated all of this reviewer’s suggestions and we think this has improved the manuscript and made it easier to follow.
Reviewer #2 (Public Review):
The authors used the ribosome profiling technique to study gene expression at transcriptional and translational levels in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2) using ribosome profiling. The ribosome profiling was carried out on the cells at different time points within the first 12 hours of infection, thus providing information on gene expression changes during the time of infection.
The analysis of ribosome profiling data is exceptionally detailed and includes scrupulous characterization of footprint read lengths, de novo prediction of translated ORFs, characterisation of local pauses and differential gene expression of host and viral genes. The RNA-seq analysis is on par with that, the authors did a superb job at characterising the composition of the viral transcriptome that included identification of heteroclite RNAs and defective interfering RNAs. This provided the authors with reliable information for the interpretation of translational mechanisms responsible for the translation of ORFs discovered with ribosome profiling data.
A specific focus of the manuscript was placed on the characterisation of two instances of ribosomal frameshifting occurring in PRRSVs. In addition to "canonical" -1 frameshifting at a slippery sequence stimulated by downstream RNA secondary structure (common to many viruses), PRRSVs genome contains an additional frameshifting site whose efficiency is stimulated by a viral protein. The authors demonstrated that the efficiency of this frameshifting is increasing over time which is expected since the concentration of stimulating protein is increasing. Furthermore, the authors found that the efficiency of "canonical" frameshifting is also changed. The authors describe this as surprising since it directly contradicts the common description of its function as "setting the fixed ratio" between the synthesized products upstream and downstream of the frameshift site. Perhaps it is not so surprising in the hindsight, given that the frameshifting is dependent on so many different factors, folding states of RNA pseudoknots which are dynamic, ribosome density upstream, etc. it would be more surprising if the efficiency of frameshifting were indeed fixed. I think the "fixed ratio" was proposed mainly to draw a difference to ribosomal frameshifting occurring in cellular genes (like antizyme or bacterial release factor 2) where there seems to be only one functional product, but its synthesis level depends on the efficiency of frameshifting sensing certain conditions. It is great though that the authors observed such changes and I agree with the authors' speculations that this is unlikely to be unique to PRRSVs.
While I found the work to be largely descriptive, the authors did not shy away from speculating about potential mechanisms responsible for observed regulation. The manuscript is hard to get through simply due to its large length and a lot of data, but reading it is rewarding.
Again, we would like to thank this reviewer for their positive comments about the work, and to reiterate that hopefully the revised version of the manuscript will be easier to read.
Reviewer #3 (Public Review):
The manuscript by Cook et al. describes the first comprehensive gene expression analysis of two species of PRRSV, an important agricultural pathogen. Using ribosome profiling and RNA-sequencing, the authors systematically analyze the transcriptome of the virus and its translation, and their temporal kinetics. The analysis revealed non-canonical RNA species that are suggested to contribute to translation of parts of ORF1ab, changing the stoichiometry between the NSPs. In addition, the authors use the ribosome profiling data to identify novel overlapping ORFs, including a conserved uORF in the 5' leader, and to analyze the efficiency of frame-shift in two sites in the viral genome, one of which is trans-regulated by the viral nsp1β. The frame-shift efficiency in both sites is presented to be increasing late in infection. The authors also present conservation analysis from hundreds of available genomes. Finally, analysis of host gene expression uncovers a pattern suggesting translation inhibition of induced transcripts, and by comparing a WT virus to a mutant virus lacking the nsp2 site frame-shift, the authors identify a gene (TXNIP) whose expression is affected by nsp2TF.
In this rigorous work, the authors uncover new insights on an important pathogen, which can be of value to the wider field of virology. However, due to technical issues a few of the authors claims may require reconsideration.
We are grateful to this reviewer for their comments on the rigour and the impact of the work, as well as the suggestions for improvement which they included in their more detailed review. Within the detailed review, this reviewer expressed some concerns that ribosome run-off (seen in Figure 1—figure supplement 1 [formerly Supplementary Figure 1]) might confound the comparison of ribosome densities in different regions of the viral genome (particularly ORF1ab). However, this run-off only noticeably affects the first ~100 nt of host CDSs, which is very small compared to the ~12,000 nt total length of ORF1ab. The regions of ORF1ab in which we compare ribosome density in our study are almost all > 1,000 nt downstream of this ~100 nt run-off region and will therefore not be significantly affected by run-off. The exception to this is our assessment of heteroclite sgRNA translation, where the “heteroclite” region does include the first ~100 nt of ORF1a. As such, run-off may have a slight effect on this analysis, but we expect this to be minor, as the ~100 nt run-off region represents only a small proportion of the 1,550-nt “heteroclite” region. Further, any such effect would actually lead to under-estimation of heteroclite sgRNA translation, by artefactually reducing the relative RPF density in the heteroclite region. This would therefore strengthen our conclusion that our data provide evidence for heteroclite sgRNA translation.
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Evaluation Summary:
The paper presents the first comprehensive gene expression analysis of two species of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes economically important diseases of pigs. By applying ribosome profiling to arterivirus-infected cells the authors discovered a substantial number of efficiently translated open reading frames in viral RNAs and obtained compelling evidence of changes in ribosome frameshifting efficiencies over the course of infection. The paper is of interest to virologists and researchers studying mRNA decoding, recoding, and translation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the …
Evaluation Summary:
The paper presents the first comprehensive gene expression analysis of two species of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes economically important diseases of pigs. By applying ribosome profiling to arterivirus-infected cells the authors discovered a substantial number of efficiently translated open reading frames in viral RNAs and obtained compelling evidence of changes in ribosome frameshifting efficiencies over the course of infection. The paper is of interest to virologists and researchers studying mRNA decoding, recoding, and translation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors use ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) to characterise the transcriptome and translatome of two PRRSV species as well as the host in response to infection. One particularly exciting feature of the study is that the analysis is carried out at different times of infection, which shows how both the virus and the host regulate their gene expression. The authors identify several new regulatory mechanisms of virus gene expression. Unexpectedly, they also find that the frameshifting efficiency at the ORF1ab frameshifting site changes with time. This contradicts the dogma in the field, which states that frameshifting is constant and has evolved to be constant to produce the a particular ratio of the two protein isoforms. The strength of the paper is in its comprehensible analysis. …
Reviewer #1 (Public Review):
The authors use ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) to characterise the transcriptome and translatome of two PRRSV species as well as the host in response to infection. One particularly exciting feature of the study is that the analysis is carried out at different times of infection, which shows how both the virus and the host regulate their gene expression. The authors identify several new regulatory mechanisms of virus gene expression. Unexpectedly, they also find that the frameshifting efficiency at the ORF1ab frameshifting site changes with time. This contradicts the dogma in the field, which states that frameshifting is constant and has evolved to be constant to produce the a particular ratio of the two protein isoforms. The strength of the paper is in its comprehensible analysis. The paper is extremely rich in data, with 12 main and 23 Supplemental Figs and 11 Supplemental Tables, all of them rather complex. The main weakness is that it is written in a technical language that will be hardly readable by a non-specialist readership. Unfortunately, the authors do not make a good job in guiding the reader through their findings and hardly identify the the most important findings, while leaving the details to the specialists. This is particularly exemplified in Fig. 12, which should present the summary of the findings and would be extremely helpful, but hardly provides any text at all. This is potentially a very interesting paper, but the impact on the field could be increased considerably by better presentation of the work.
-
Reviewer #2 (Public Review):
The authors used the ribosome profiling technique to study gene expression at transcriptional and translational levels in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2) using ribosome profiling. The ribosome profiling was carried out on the cells at different time points within the first 12 hours of infection, thus providing information on gene expression changes during the time of infection.
The analysis of ribosome profiling data is exceptionally detailed and includes scrupulous characterization of footprint read lengths, de novo prediction of translated ORFs, characterisation of local pauses and differential gene expression of host and viral genes. The RNA-seq analysis is on par with that, the authors did a superb job at characterising the composition of …
Reviewer #2 (Public Review):
The authors used the ribosome profiling technique to study gene expression at transcriptional and translational levels in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2) using ribosome profiling. The ribosome profiling was carried out on the cells at different time points within the first 12 hours of infection, thus providing information on gene expression changes during the time of infection.
The analysis of ribosome profiling data is exceptionally detailed and includes scrupulous characterization of footprint read lengths, de novo prediction of translated ORFs, characterisation of local pauses and differential gene expression of host and viral genes. The RNA-seq analysis is on par with that, the authors did a superb job at characterising the composition of the viral transcriptome that included identification of heteroclite RNAs and defective interfering RNAs. This provided the authors with reliable information for the interpretation of translational mechanisms responsible for the translation of ORFs discovered with ribosome profiling data.
A specific focus of the manuscript was placed on the characterisation of two instances of ribosomal frameshifting occurring in PRRSVs. In addition to "canonical" -1 frameshifting at a slippery sequence stimulated by downstream RNA secondary structure (common to many viruses), PRRSVs genome contains an additional frameshifting site whose efficiency is stimulated by a viral protein. The authors demonstrated that the efficiency of this frameshifting is increasing over time which is expected since the concentration of stimulating protein is increasing. Furthermore, the authors found that the efficiency of "canonical" frameshifting is also changed. The authors describe this as surprising since it directly contradicts the common description of its function as "setting the fixed ratio" between the synthesized products upstream and downstream of the frameshift site. Perhaps it is not so surprising in the hindsight, given that the frameshifting is dependent on so many different factors, folding states of RNA pseudoknots which are dynamic, ribosome density upstream, etc. it would be more surprising if the efficiency of frameshifting were indeed fixed. I think the "fixed ratio" was proposed mainly to draw a difference to ribosomal frameshifting occurring in cellular genes (like antizyme or bacterial release factor 2) where there seems to be only one functional product, but its synthesis level depends on the efficiency of frameshifting sensing certain conditions. It is great though that the authors observed such changes and I agree with the authors' speculations that this is unlikely to be unique to PRRSVs.
While I found the work to be largely descriptive, the authors did not shy away from speculating about potential mechanisms responsible for observed regulation. The manuscript is hard to get through simply due to its large length and a lot of data, but reading it is rewarding.
-
Reviewer #3 (Public Review):
The manuscript by Cook et al. describes the first comprehensive gene expression analysis of two species of PRRSV, an important agricultural pathogen. Using ribosome profiling and RNA-sequencing, the authors systematically analyze the transcriptome of the virus and its translation, and their temporal kinetics. The analysis revealed non-canonical RNA species that are suggested to contribute to translation of parts of ORF1ab, changing the stoichiometry between the NSPs. In addition, the authors use the ribosome profiling data to identify novel overlapping ORFs, including a conserved uORF in the 5' leader, and to analyze the efficiency of frame-shift in two sites in the viral genome, one of which is trans-regulated by the viral nsp1β. The frame-shift efficiency in both sites is presented to be increasing late in …
Reviewer #3 (Public Review):
The manuscript by Cook et al. describes the first comprehensive gene expression analysis of two species of PRRSV, an important agricultural pathogen. Using ribosome profiling and RNA-sequencing, the authors systematically analyze the transcriptome of the virus and its translation, and their temporal kinetics. The analysis revealed non-canonical RNA species that are suggested to contribute to translation of parts of ORF1ab, changing the stoichiometry between the NSPs. In addition, the authors use the ribosome profiling data to identify novel overlapping ORFs, including a conserved uORF in the 5' leader, and to analyze the efficiency of frame-shift in two sites in the viral genome, one of which is trans-regulated by the viral nsp1β. The frame-shift efficiency in both sites is presented to be increasing late in infection. The authors also present conservation analysis from hundreds of available genomes. Finally, analysis of host gene expression uncovers a pattern suggesting translation inhibition of induced transcripts, and by comparing a WT virus to a mutant virus lacking the nsp2 site frame-shift, the authors identify a gene (TXNIP) whose expression is affected by nsp2TF.
In this rigorous work, the authors uncover new insights on an important pathogen, which can be of value to the wider field of virology. However, due to technical issues a few of the authors claims may require reconsideration.
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