RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies

Thank you for sending your work entitled “RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies” for consideration at eLife. Your article has been favorably evaluated by Richard Losick (Senior editor), a Reviewing editor, and 3 reviewers, one of whom, Raul Andino, has agreed to reveal his identity.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. The comments in full from the three reviewers are attached below.

Our reviewers were in close agreement that the paper makes important points and is topical and appropriate for eLife. There was also a consensus that there were some failings in reviewing the history of this topic, and I agree that an improved review is needed (apparently key citations are missing). This should be easily fixed. There were some good suggestions to improve clarity. There were also requests for more experimentation from one (the third) reviewer, which I think sound reasonable, but I cannot evaluate how much work is involved. I would encourage the authors to consider doing what is easy. It seems to this reviewer that the quantitation might be improved without huge effort. We are willing to leave it to the authors to decide how much more wet work is desired.

All told, we are supportive of publication.

Reviewer #1

This manuscript presents a careful and interesting work, and even more thoroughly documents on the attenuating effect of dinucleotides CpG and UpA in the genomes of RNA viruses than previous papers have. They argue, convincingly, that attenuating effects previously ascribed to 'codon pair bias' in attenuating viruses can be completely explained by the resulting increases in CpG and UpA dinucleotides. They do this via a systematic analysis of the attenuating effects of altering 'codon-pair bias' in previous papers and by carefully design experiments of their own, which include documentation that the attenuation does not occur by reducing translational efficiency, but by reducing the infectivity of the resulting virions.

With respect to the possible attenuation mechanism of CpG and UpA dinucleotides, the authors discuss the possibility that it is a mechanism of the innate immune response that is not PKR- or pattern receptor-dependent. Data relevant to this are published separately (Atkinson et al., 2014).

The curious thing about this field, and this manuscript, is that the 'straw man' of codon-pair bias was already destroyed by Burns et al., in 2009. These authors were pursuing their own hypothesis, that the substitution of rare codons would attenuate viruses. They found that this was the case, but careful mutagenesis revealed that it was, in fact, the increase in CpG and UpA nucleotides in the RNA that accounted for the attenuation. They showed that this effect was not due to translational efficiency and was manifested as the increased particle-to-PFU ratio of the resulting viruses. They reflected that bias against CpG and UpA codons might be the reason, in fact, that codons that contain them, and codon pairs that create them, are rare to begin with.

They noted, quite politely, that this also accounted for the effect of the highly publicized “codon-pair bias” published by Coleman et al., 2008. They stated: “A prominent feature of the most disfavored codon pairs is the presence of CpG or UpA across codons. Thus, the observed CPB in poliovirus and in humans and higher eukaryotes may be driven primarily by CpG and UpA dinucleotide suppression. In this context, it is notable that in cassette C of the construct with the lowest fitness, ABc₁₂, within-codon CpG and UpA frequencies were maximized but the CPB score was similar to those of higher-fitness constructs, including ABC.” Thus, like the current manuscript, they re-analyzed the data of Coleman et al., and found it to be completely explicable in terms of CpG and UpA dinucleotides.

This conclusion, and the published data, were clearly insufficient to prevent the design and interpretation of several more manuscripts using 'codon pair bias' to design attenuated viruses for vaccine and basic science purposes (Mueller et al., 2010; Martrus, 2013; Yang, 2013; Ni, 2014).

In short, this is a well-executed, important study that extends the very clear conclusions of the also excellent, but completely ignored, 2009 paper of Burns et al. Therefore its publication would bring an important discussion into the limelight. Here are my suggestions, mostly having to do with writing and scholarship:

1) That CpG and UpA dinucleotide frequency correlates with attenuation of viruses, rather than 'codon pair bias' or 'codon de-optimization', is a feature of these approaches rather than an artifact, in this reviewer's opinion. The combative nature of the title is not necessary to bring this fact to light.

2) It does not detract from the quality of the present work to acknowledge more explicitly the contributions of Burns et al. It may, however, detract from its novelty.

3) The readers understanding of the data is compromised by the poor description of the experiments and analysis in the figure legends. These should be rewritten in such a way that the figures can be independently understood. For example, nowhere in the manuscript can the metric for “CPB score” be discovered.

4) Similarly, the quantitative findings in Figure 4 are difficult to dissect. With so many changes in the genomes being analyzed, qRT-PCR of viral competitions would be helpful. As it is, the actual data from the competition experiments should be presented, rather than a cartoon depicting which viruses 'won' and 'lost'.

5) The last paragraph of the Discussion uses 'data not shown' to make its point. These data should be shown or the sentences deleted.

Reviewer #2

There has been interesting discussions on why the nucleotide composition of many human viruses present distinct nucleotide, di-nucleotide and codon preferences. In the last years synthetic viruses have been generated with alternative codon distributions that show attenuated replication. However these constructs change other variables (nucleotide or dinucleotide composition) in addition to the codon distribution and it is unclear what of those changes affects replication. The authors address this issue by generating mutants of the echovirus 7 in which CpG and UpA dinucleotides were varied independent of the codon distribution. The results clearly showed that the main factor dominating replication was dinucleotide content, and that translation efficiency was unaffected by the two variables. The authors then argue that attenuation is mediated through innate immune response to viruses with high CpG/UpA content.

The paper is very interesting and results are compelling. The main critiques are:

1) The topic of dinucleotide biases in RNA viruses has been explored extensively and most references are absent.

2) The discussion on the mechanisms of attenuation and the association to the innate immune response is not well elaborated. They mention the lack of association to interferon response and the effect of C16. However, the results are not presented in detail. I suggest the authors to strengthen their conclusions.

Reviewer #3

This study examines the role of nucleotide composition in RNA virus genomes. The authors introduced a number of synonymous mutations into the EV71 genome to modify the frequency of CpG and UpA dinucleotide (DN) or codon pair (CP). Their results support the idea that DN frequencies determine virus replication, while neither DN nor CP affected translation efficiency. They concluded that DN affect virus replication because the nucleotide composition of the virus genome influences the host-cell innate response to the virus.

The most interesting contributions of this study are: 1) they are able to identify mutations that will affect CP without affecting CpG/UpA dinucleotide composition and vice versa; (Bennetzen and Hall, 1982) based on this bioinformatics information they constructed mutants that change CP or DN composition and experimentally evaluated the effect of synonymous mutation on virus replication. Their conclusions are supported by the available results, and I think this study represents an important contribution to the field because it seems to address a molecular mechanism for virus genome nucleotide composition bias. However, I believe that a more quantitative analysis of the competition experiment may be required to determine the degree of correlation between DN composition and virus replication fitness.

The authors choose competition assays to precisely analyze fitness between different virus and with respect to WT. This is the correct experiment in my opinion, however I believe the analysis of the experiment is somewhat casual and not very quantitative, and therefore limits the value of this data. Figure 4 presents the results using a differential restriction enzyme pattern to distinguish between the two competing viruses, but they can only determine when one virus is lost (no longer detected by the assay). I think that it will be a lot more powerful to use digital PCR to precisely quantify the ratio between virus genomes in the given competition assay. This will provide parameters that can then be fit in a simple mathematical model to determine with more accuracy the correlation between DN or CP and fitness, which at this point seems a bit circumstantial.

Similarly, it would be desirable to improve the quality of the in vitro translation assay and quantify protein production to determine that, in this case, there is little correlation between translation efficiency and fitness, as this is one of the central claims of the study.

Reviewer #1

1) That CpG and UpA dinucleotide frequency correlates with attenuation of viruses, rather than 'codon pair bias' or 'codon de-optimization', is a feature of these approaches rather than an artifact, in this reviewer's opinion. The combative nature of the title is not necessary to bring this fact to light.

As co-authors, we did spend some time discussing the title of the paper. The final choice was in fact motivated for reasons alluded to by the reviewer: that those involved in the codon pair programme have simply ignored the published evidence, by Burns et al. and more recently from our lab (Atkinson et al.), that the attenuating effect was mediated through inadvertently increasing CpG ad UpA dinucleotide frequencies rather any effect on translation. A clear, declarative title of the paper seems required to counter mistaken views on this. If we changed the title as the reviewer suggests, it would imply that codon pair de-optimisation has been used deliberately as a way to increase CpG and UpA dinucleotide frequencies. This is absolutely not the case.

That said, we would have liked to qualify that statement by stating why we make this assertion, but the low character limit imposed on eLife papers titles prevents us from doing this.

2) It does not detract from the quality of the present work to acknowledge more explicitly the contributions of Burns et al. It may, however, detract from its novelty.

We have cited that study in the original manuscript. In the Introduction of the revised manuscript, we have described the observation made by Burns and colleagues in more detail as requested by the reviewer.

3) The readers understanding of the data is compromised by the poor description of the experiments and analysis in the figure legends. These should be rewritten in such a way that the figures can be independently understood. For example, nowhere in the manuscript can the metric for “CPB score” be discovered.

We apologise for these omissions and have endeavoured to make the figure legends clearer (more explanatory) and describe the various abbreviations more fully. We have dropped the abbreviations CPB as it can be expressed more clearly by other wordings (e.g. biased codon pair usage).

4) Similarly, the quantitative findings in Figure 4 are difficult to dissect. With so many changes in the genomes being analyzed, qRT-PCR of viral competitions would be helpful. As it is, the actual data from the competition experiments should be presented, rather than a cartoon depicting which viruses 'won' and 'lost'.

We fully agree that the results presentation in Figure 4B was unduly diagrammatic and lacked primary data (with the exception of the two example gel images in Figure 4A). This point was also made by Reviewer #3. To address this, we have made use of the possibility to include figure supplements by now including gel images of the other competition assays so that relative fitness can be directly evaluated. We have, however, retained the original Figure 4B as a summary of the experimental data.

The use of restriction enzymes to differentiate E7 mutants is a widely used method and its results can be made fully quantitative using appropriately calibrated controls. Had the phenotypes been more subtle we agree that quantitative qRT-PCR would likely have been necessary to discriminate between the fitness of viruses with modified dinucleotide ratios. However, in the current study, outcomes were either elimination of one or other of the competing viruses or a draw. These results are readily visualised as presented, and we believe the investigation does not need more precise quantitation as suggested by the reviewer.

5) The last paragraph of the discussion uses 'data not shown' to make its point. These data should be shown or the sentences deleted.

We agree, and that part of the discussion has entirely been removed. We have also removed reference to unpublished investigations of Theiler’s virus and influenza A virus from the Introduction.

Reviewer #2

1) The topic of dinucleotide biases in RNA viruses has been explored extensively and most references are absent.

We have now cited, in the Introduction, the Rima and Karlin studies that originally noted the suppression of CpG and UpA dinucleotide frequencies in RNA viruses.

2) The discussion on the mechanisms of attenuation and the association to the innate immune response is not well elaborated. They mention the lack of association to interferon response and the effect of C16. However, the results are not presented in detail. I suggest the authors to strengthen their conclusions.

That part of the Discussion was based on the results presented in the Atkinson et al. paper from earlier in the year. We have modified that paragraph to clarify that we were referring to this previous study.

Reviewer #3

This study examines the role of nucleotide composition in RNA virus genomes. The authors introduced a number of synonymous mutations into the EV71 genome to modify the frequency of CpG and UpA dinucleotide (DN) or codon pair (CP). Their results support the idea that DN frequencies determine virus replication, while neither DN nor CP affected translation efficiency. They concluded that DN affect virus replication because the nucleotide composition of the virus genome influences the host-cell innate response to the virus.

The most interesting contributions of this study are: 1) they are able to identify mutations that will affect CP without affecting CpG/UpA dinucleotide composition and vice versa; (Bennetzen and Hall, 1982) based on this bioinformatics information they constructed mutants that change CP or DN composition and experimentally evaluated the effect of synonymous mutation on virus replication. Their conclusions are supported by the available results, and I think this study represents an important contribution to the field because it seems to address a molecular mechanism for virus genome nucleotide composition bias. However, I believe that a more quantitative analysis of the competition experiment may be required to determine the degree of correlation between DN composition and virus replication fitness.

The authors choose competition assays to precisely analyze fitness between different virus and with respect to WT. This is the correct experiment in my opinion, however I believe the analysis of the experiment is somewhat casual and not very quantitative, and therefore limits the value of this data. Figure 4 presents the results using a differential restriction enzyme pattern to distinguish between the two competing viruses, but they can only determine when one virus is lost (no longer detected by the assay). I think that it will be a lot more powerful to use digital PCR to precisely quantify the ratio between virus genomes in the given competition assay. This will provide parameters that can then be fit in a simple mathematical model to determine with more accuracy the correlation between DN or CP and fitness, which at this point seems a bit circumstantial.

This comment is related to that of Reviewer #1 and has been addressed above (the limited number of observed outcomes of competition assays can be effectively demonstrated through restriction enzyme analysis).

To address the issue of relative fitness further, we have repeated the experiment to determine the replication kinetics of WT and mutants of E7 that was depicted in Figure 3. The re-formatting of data as histograms with error bars now allows replication rates from all mutants at different time points to be shown and these entirely back up the competition assay results.

Similarly, it would be desirable to improve the quality of the in vitro translation assay and quantify protein production to determine that, in this case, there is little correlation between translation efficiency and fitness, as this is one of the central claims of the study.

We did indeed quantify translation of the individual proteins on the blot by densitometry and presented the results in the form of a histogram in Figure 5–figure supplement 1. We additionally used a translation efficiency metric based on this quantitation to analyse potential associations with virus replication rate (Table 4) along with other variables (dinucleotide composition, CB bias, CAI, ENc and G + C content).