Reply to Simmonds et al.: Codon pair and dinucleotide bias have not been functionally distinguished
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Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans the biological, physical, and social sciences.
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Written from the paper’s full text.
TL;DR
This reply argues that codon pair bias and CpG/UpA dinucleotide bias have not been functionally distinguished in the criticized work or in Simmonds et al.’s Min-H virus. It contends that recoding changes both features coordinately, that Min-H’s average codon pair score masks increased disfavored pairs, and that the vaccine-safety concern is premature.
Key findings
- Futcher et al. argue that UpA/CpG dinucleotides available for change are largely at codon–codon junctions, where they define codon pairs; in mammals, many disfavored codon pairs have UpA or CpG at their junctions, so altering one alters the other. All viral constructs in question coordinately alter dinucleotides and codon pairs and therefore do not distinguish the phenomena.
- They dispute Simmonds et al.’s claim that Min-H has the same codon pair frequencies as control Min-E: in Min-H, CpG/UpA and disfavored codon pairs are coordinately increased (Figs. 1 and 2). They say positive-scoring codon pairs were introduced elsewhere to keep the average codon pair score the same, masking increased poor pairs; equal averages do not mean equal frequencies.
- They state that no mechanism is known for either phenomenon, and that their results, conclusions, or future uses would not change if the viruses were called “dinucleotide deoptimized.” They also say they do not claim codon pair bias necessarily works through translation; cell-line variation is common, and cultured-cell versus animal properties can differ.
- They call concern about future codon pair vaccines in immunocompromised individuals premature because live vaccines are generally contraindicated and extending tissue culture/mouse experiments to humans would require lengthy studies.
Methods
- This is a reply/commentary; no new experimental design, sample, or statistical analysis is reported.
- The argument relies on interpretation of previously published viral constructs, including Min-H, Min-E, and Min-U, and cited figures (Figs. 1 and 2).
- No quantitative data or new analyses are presented in the text.
Limitations
- Stated: no mechanism is known for either codon pair or dinucleotide effects; properties in cultured cells and animals can be very different; extending tissue culture/mouse experiments to humans would involve lengthy studies.
- Stated: the authors report a conflict of interest: E.W. is a co-founder and C.B.S. and S.M. are employees of Codagenix Inc.
- Evident: as a reply, it does not provide new experiments to functionally separate codon pair and dinucleotide bias; it argues that the existing constructs confound the two.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
6 of 6 cited sources checked · 3 supported · 2 flagged · accuracy not yet measured
- Interpretationreasoning
All of the viral constructs in question (from Simmonds et al. and Shen et al.) coordinately alter dinucleotides and codon pairs.
“Accordingly, all of the viral constructs in question (1, 2) coordinately alter dinucleotides and codon pairs (see below)”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [1] Simmonds P, Tulloch F, Evans DJ, Ryan MD. Attenuation of dengue (and other RNA viruses) with codon pair recoding can be explained by increased CpG/UpA dinucleotide frequencies. Proc Natl Acad Sci USA. 2015;112:E3633–E3634. doi: 10.1073/pnas.1507339112. [DOI] [PMC free article] [PubMed] [Google Scholar] source
The paper says Shen et al.’s three recoded dengue regions changed both codon-pair use and CpG/UpA frequencies. But its discussion of the “Min” mutants gives an explicit exception to the claim about all constructs: Min-E incorporated disfavored codon pairs while keeping CpG and UpA frequencies at wild-type levels. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured - cites [2] Shen SH, et al. Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference. Proc Natl Acad Sci USA. 2015;112(15):4749–4754. doi: 10.1073/pnas.1502864112. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Findingcitation
Tulloch et al. introduced positive-scoring codon pairs at other positions in Min-H, producing the same average codon pair score as the control.
“Tulloch et al. (3) introduced positive-scoring codon pairs at other positions (Fig. 1), producing the same average codon pair score as the control.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [3] Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. 2014;3:e04531. doi: 10.7554/eLife.04531. [DOI] [PMC free article] [PubMed] [Google Scholar] source
The Results section (‘Separate assessment of effects of CP and dinucleotide frequencies on virus replication’; page unavailable) says Min-H was generated with the same codon-pair frequencies as Min-E, not with the same average score as the WT or permuted control. It reports a score of −0.111 for Min-E versus −0.014 for WT. The cited passage also does not describe adding positive-scoring codon pairs at other positions. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
- Premisecitation
In mammals, many disfavored codon pairs have UpA or CpG at their junctions.
“In mammals, many disfavored codon pairs have UpA or CpG at their junctions (4).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [4] Coleman JR, et al. Virus attenuation by genome-scale changes in codon pair bias. Science. 2008;320(5884):1784–1787. doi: 10.1126/science.1155761. [DOI] [PMC free article] [PubMed] [Google Scholar] source
The passage labeled Abstract gives a human example of an underrepresented codon pair, GCC|GAA, which has CpG at the codon junction. It does not say that many disfavored pairs have CpG junctions or mention UpA junctions, so it supports only the narrower example. (LLM, conf=0.84)
Experimental — accuracy not yet measured
- Findingcitation
Our recoded viruses have increased CpG/UpA.
“the authors suggest that our recoded viruses have increased CpG/UpA. Yes, this is necessarily true (3).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [3] Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife. 2014;3:e04531. doi: 10.7554/eLife.04531. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Premisecitation
Properties of a virus variant in cultured cells and animals can be very different.
“Properties of a virus variant in cultured cells and animals can be very different (2).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [2] Shen SH, et al. Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference. Proc Natl Acad Sci USA. 2015;112(15):4749–4754. doi: 10.1073/pnas.1502864112. [DOI] [PMC free article] [PubMed] [Google Scholar] source
- Findingown data
Min-H does not have the same codon pair frequencies as control Min-E.
“it is incorrect to describe the virus as having the same codon pair frequencies as Min-E”
Quote in the paperSubstantial claim - Findingown data
In Min-H, frequencies of CpG/UpA and disfavored codon pairs are coordinately increased.
“In fact, in Min-H, frequencies of CpG/UpA and disfavored codon pairs are coordinately increased (Figs. 1 and 2)”
- Also stated in: another passage · plain
“to the contrary, frequencies of poor pairs have been increased coordinately with CpG/UpA dinucleotides”
Quote in the paperSubstantial claim - Also stated in: another passage · plain
- Interpretationown data
In Min-H, CpG/UpA and disfavored codon pair frequencies are not distinguished.
“and thus not distinguished”
Quote in the paperSubstantial claim - Interpretationreasoning
These viral constructs do not distinguish the phenomena of dinucleotide bias and codon pair bias.
“and so do not distinguish the phenomena”
Quote in the paperSubstantial claim
- May combine two claims. 2
- Classified as probably not a substantial claim. 1
May combine two claims.
InterpretationreasoningThese viruses (Min-H and Min-U) do not distinguish the phenomena, if the phenomena are even different.
“Thus, these viruses do not distinguish the phenomena, if the phenomena are even different.”
Quote in the paperSubstantial claim- Rests on an assumption: There is no reason to think the attenuating effects of the many disfavored codon pairs in Min-H (or Min-U) can be suppressed or balanced by adding positive-scoring codon pairs elsewhere.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).
Interpretationnone givenExtending our tissue culture/mouse experiments to humans would involve lengthy studies.
“Extending our tissue culture/mouse experiments to humans would involve lengthy studies”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningAltering UpA/CpG dinucleotides simultaneously alters codon pairs, and vice versa.
“Therefore, altering UpA/CpG dinucleotides simultaneously alters codon pairs, and vice versa.”
Quote in the paperSubstantial claim- Rests on an assumption: When a protein is recoded, UpA/CpG dinucleotides available for change are largely at codon–codon junctions, where they define codon pairs.
The paper relies on this without support.
When a protein is recoded, UpA/CpG dinucleotides available for change are largely at codon–codon junctions, where they define codon pairs.
“When a protein is recoded, UpA/CpG dinucleotides available for change are largely at codon–codon junctions, where they define codon pairs.”
The paper relies on this without support.
Live vaccines are generally contraindicated in immunocompromised individuals.
“live vaccines are generally contraindicated in such circumstances, a fact already widely appreciated”
The paper relies on this without support.
Variation between mammalian cell lines is seen for virtually all phenomena.
“Variation between mammalian cell lines is seen for virtually all phenomena.”
The paper relies on this without support.
There is no reason to think the attenuating effects of the many disfavored codon pairs in Min-H (or Min-U) can be suppressed or balanced by adding positive-scoring codon pairs elsewhere.
“There is no reason to think the attenuating effects of the many disfavored codon pairs in Min-H (or Min-U) can be suppressed or balanced by adding positive-scoring codon pairs elsewhere.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The molecular mechanisms that underlie the attenuation by CPD have remained unknown.
“Despite its great potential, the molecular mechanisms that underlie the attenuation by CPD have remained unknown (Broadbent et al., 2016;Coleman et al., 2008;Futcher et al., 2015;Le Noue ¨n et al., 2014;Shen et al., 2015;Simmonds et al., 2015;Yang et al., 2013).”
From Mechanism of Virus Attenuation by Codon Pair Deoptimization (2020) · cites this paper as Futcher et al., 2015
Cites
Virus attenuation by genome-scale changes in codon pair bias
Coleman, J. R., Papamichail, D., Skiena, S., Futcher, B., Wimmer, E., & Mueller, S. (2008) · Science · doi:10.1126/science.1155761 · Cited by 715 · see citing works
Self-citationTulloch, F., Atkinson, N. J., Evans, D. J., Ryan, M. D., & Simmonds, P. (2014) · eLife · doi:10.7554/elife.04531 · Cited by 178 · see citing works
Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference
Shen, S. H., Stauft, C. B., Gorbatsevych, O., Song, Y., Ward, C. B., Yurovsky, A., Mueller, S., Futcher, B., & Wimmer, E. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1502864112 · Cited by 99 · see citing works
Self-citationSimmonds, P., Tulloch, F., Evans, D. J., & Ryan, M. D. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1507339112 · Cited by 28 · see citing works
Flagged first, then by citations
Cited by
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mRNA structure regulates protein expression through changes in functional half-life
Mauger, D. M., Cabral, B. J., Presnyak, V., Su, S. V., Reid, D. W., Goodman, B., Link, K., Khatwani, N., Reynders, J., Moore, M. J., et al. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1908052116 · Cited by 555 · see citing works
“Because these effects are interdependent on mRNA sequence, teasing apart their individual contributions to protein output is difficult and often controversial (7, 8).”
BackgroundCG dinucleotide suppression enables antiviral defence targeting non-self RNA
Takata, M. A., Gonçalves-Carneiro, D., Zang, T. M., Soll, S. J., York, A., Blanco-Melo, D., & Bieniasz, P. D. (2017) · Nature · doi:10.1038/nature24039 · Cited by 398 · see citing works
Synonymous Codons: Choose Wisely for Expression
Brule, C. E., & Grayhack, E. J. (2017) · Trends in Genetics · doi:10.1016/j.tig.2017.02.001 · Cited by 231 · see citing works
“[83] suggests that use of such pairs is deleterious to expression although the nature of their effects remains to be deciphered [84, 85].”
BackgroundAlexaki, A., Kames, J. M., Holcomb, D. D., Athey, J. C., Santana-Quintero, L. V., Lam, P. V., Hamasaki‐Katagiri, N., Osipova, E., Simonyan, V. V., Bar, H., et al. (2019) · Journal of Molecular Biology · doi:10.1016/j.jmb.2019.04.021 · Cited by 180 · see citing works
Odon, V., Fros, J. J., Goonawardane, N., Dietrich, I., Ibrahim, A., Alshaikhahmed, K., Nguyen, D. V., & Peter Simmonds, K. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz581 · Cited by 93 · see citing works
Mechanism of Virus Attenuation by Codon Pair Deoptimization
Groenke, N., Trimpert, J., Merz, S., Conradie, A. M., Wyler, E., Zhang, H., Hazapis, O.-G., Rausch, S., Landthaler, M., Osterrieder, N., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2020.107586 · Cited by 85 · see citing works
“Hence, uncoupling the effects of the two phenomena is difficult (Broadbent et al., 2016; Futcher et al., 2015; Simmonds et al., 2015).”
BackgroundSynonymous Virus Genome Recoding as a Tool to Impact Viral Fitness
Martı́nez, M. Á., Jordan-Paiz, A., Franco, S., & Nevot, M. (2015) · Trends in Microbiology · doi:10.1016/j.tim.2015.11.002 · Cited by 81 · see citing works
“The attenuation mechanism of synonymously deoptimized virus genomes is controversial [68,69].”
BackgroundZika Virus Attenuation by Codon Pair Deoptimization Induces Sterilizing Immunity in Mouse Models
Li, P., Ke, X., Wang, T., Tan, Z., Luo, D., Miao, Y., Sun, J., Zhang, Y., Liu, Y., Hu, Q., et al. (2018) · Journal of Virology · doi:10.1128/jvi.00701-18 · Cited by 74 · see citing works
“Codon pair bias has been suggested to be a direct consequence of CpG/UpA dinucleotide bias (55), and the increased frequency of CpG/UpA dinucleotides may be a key genetic contributor to virus attenuation by codon pair deoptimization (56), although this has been disputed (28, 57).”
BackgroundFros, J. J., Dietrich, I., Alshaikhahmed, K., Passchier, T. C., Evans, D. J., & Peter Simmonds, K. (2017) · eLife · doi:10.7554/elife.29112 · Cited by 58 · see citing works
“, 2014), although this conclusion has been disputed (Futcher et al., 2015).”
BackgroundSegundo, F. D.-S., Medina, G. N., Ramírez-Medina, E., Velázquez-Salinas, L., Koster, M. J., Grubman, M. J., & Santos, T. D. L. (2015) · Journal of Virology · doi:10.1128/jvi.02167-15 · Cited by 48 · see citing works
“Comparative analysis of the viruses used in our studies showed that the A12P1 deopt virus had an increased dinucleotide frequency, and this might be one possible reason for the observed attenuation; however, at this point, codon pair and dinucleotide biases have not been functionally distinguished, and the topic remai…”
MethodIntra-genome variability in the dinucleotide composition of SARS-CoV-2
Digard, P., Lee, H. M., Sharp, C., Grey, F. E., & Gaunt, E. R. (2020) · Virus Evolution · doi:10.1093/ve/veaa057 · Cited by 41 · see citing works
Trus, I., Udenze, D., Berube, N., Wheler, C. L., Martel, M.-J., Gerdts, V., & Karniychuk, U. U. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2019.03077 · Cited by 40 · see citing works
Compositional biases in RNA viruses: Causes, consequences and applications
Gaunt, E. R., & Digard, P. (2021) · Wiley Interdisciplinary Reviews - RNA · doi:10.1002/wrna.1679 · Cited by 34 · see citing works
Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies
Martı́nez, M. Á., Jordan-Paiz, A., Franco, S., & Nevot, M. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz831 · Cited by 18 · see citing works
“Because codon-pair bias and dinucleotide frequency are inevitably connected, whether one is generating the other is still in question (37,38,43,44).”
BackgroundΦX174 Attenuation by Whole-Genome Codon Deoptimization
Leuven, J. T. V., Ederer, M. M., Burleigh, K., Scott, L., Hughes, R. A., Codrea, V., Ellington, A. D., Wichman, H. A., & Miller, C. R. (2020) · Genome Biology and Evolution · doi:10.1093/gbe/evaa214 · Cited by 15 · see citing works
Stauft, C. B., Song, Y., Gorbatsevych, O., Pantoja, P., Rodríguez, I. V., Futcher, B., Sariol, C. A., & Wimmer, E. (2019) · Virology · doi:10.1016/j.virol.2019.09.003 · Cited by 16 · see citing works
Self-citationAttenuation of Marek's disease virus by codon pair deoptimization of a core gene
Conrad, S. J., Silva, R. F., Hearn, C. J., et al. (2018) · Virology · doi:10.1016/j.virol.2018.01.020 · Cited by 15 · see citing works
Stauft, C. B., Shen, S. H., Song, Y., Gorbatsevych, O., Asare, E., Futcher, B., Mueller, S., Payne, A. F., Brecher, M. B., Kramer, L. D., et al. (2018) · PLoS ONE · doi:10.1371/journal.pone.0198303 · Cited by 13 · see citing works
Self-citationChin, W.-X., Kong, H. Y., Zhu, I. X. Y., et al. (2023) · PLoS Pathogens · doi:10.1371/journal.ppat.1011753 · Cited by 11 · see citing works
Attenuation of Viruses by Large-Scale Recoding of their Genomes: the Selection Is Always Biased
Osterrieder, N., & Kunec, D. (2018) · Current Clinical Microbiology Reports · doi:10.1007/s40588-018-0080-3 · Cited by 10 · see citing works
Te, N., Chin, A. W. H., Gu, H.-G., Jia, J. Z., Huang, Y., Cheng, S. M. S., Tan, C.-W., Zhao, J.-C., Hui, K. P. Y., Chan, M. C. W., et al. (2026) · Proceedings of the National Academy of Sciences of the United States of America · doi:10.1073/pnas.2518645123 · Cited by 1 · see citing works
“It has been argued that codon (pair) bias is an artifact of underlying changes in CpG/UpA frequencies (35 –37) and various mechanisms, from ZAP - mediated innate sensing (38) to impacts on polymerase fidelity (39), have been proposed with different forms of evidence.”
Susma, B., Spronken, M. I. J., van Nieuwkoop, S., Kalverda, B., Gultyaev, A. P., Fouchier, R. A. M., & van den Hoogen, B. G. (2026) · Journal of Virology · doi:10.1128/jvi.00047-26
- Authors
- Futcher, B., Gorbatsevych, O., Shen, S. H., Stauft, C. B., Song, Y., Wang, B., Leatherwood, J., Gardin, J., Yurovsky, A., Mueller, S., & Wimmer, E.
- Affiliations
- Stony Brook University (US)
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2015-06-12
- Volume
- 112
- Issue
- 28
- Pages
- E3635-6
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC4507201
- OpenAlex
- W1842051142
- Semantic Scholar
- e5a0a4d38d71f4b86e74a7dd4cab715a8556bac2
- Cited by
- 22
- References
- 4
- Citations by year
- 2026: 2 · 2023: 1 · 2021: 1 · 2020: 4 · 2019: 5 · 2018: 4 · 2017: 3 · 2015: 2
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (submittedVersion)
- PubMed Central (submittedVersion)
- Funders
- HHS | NIH | National Institute of Allergy and Infectious Diseases (AI07521901)
- HHS | NIH | National Institute of General Medical Sciences (GM098400)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fractal and DNA sequence analysis
- Keywords
- codon pair bias, codon usage, translation efficiency
- MeSH terms
- Animals, Arboviruses, Humans, Insect Vectors, Mammals, Genome, Viral
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