RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies
Open accessAbstract
Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
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?
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.”
From Reply to Simmonds et al.: Codon pair and dinucleotide bias have not been functionally distinguished (2015) · cites this paper as [3]
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 measuredA study of echovirus mutants demonstrated that the increase of CpG and TpA frequencies impaired virus fitness but that alteration of CPB without changing the CpG and TpA frequency did not.
“A study that analyzed a library of echovirus mutants -in which either CpG and TpA frequency or the overall CPB was kept constant while the other feature was altered -demonstrated that the increase of CpG and TpA frequencies impaired virus fitness but that alternation of CPB without changing the CpG and TpA frequency did not (Tulloch et al., 2014).”
From Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias (2016) · cites this paper as Tulloch et al., 2014
Viruses that were engineered by SAVE might not be weakened per se but are attenuated because they induce stronger immune responses.
“This means that viruses that were engineered by SAVE might not be weakened per se but that they are attenuated because they induce stronger immune responses (Tulloch et al., 2014).”
From Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias (2016) · cites this paper as Tulloch et al., 2014
Our recoded viruses have increased CpG/UpA.
“the authors suggest that our recoded viruses have increased CpG/UpA. Yes, this is necessarily true (3).”
From Reply to Simmonds et al.: Codon pair and dinucleotide bias have not been functionally distinguished (2015) · cites this paper as [3]
Codon pair preferences and dinucleotide frequencies are tightly linked.
“As codon pair preferences and dinucleotide frequencies are tightly linked, disentangling the effects of two phenomena is exquisitely difficult (Kunec and Osterrieder, 2016;Tulloch et al., 2014).”
From Mechanism of Virus Attenuation by Codon Pair Deoptimization (2020) · cites this paper as Tulloch et al., 2014
Increasing the frequency of CpG dinucleotides in RNA virus genomes reduces virus fitness.
“Several studies have shown that increasing the frequency of CpG dinucleotides in the RNA virus genome reduces the virus fitness (Atkinson et al., 2014;Burns et al., 2009;Tulloch et al., 2014).”
From Mechanism of Virus Attenuation by Codon Pair Deoptimization (2020) · cites this paper as Tulloch et al., 2014
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Nonrandom utilization of codon pairs in Escherichia coli.
Gutman, G. A., & Hatfield, G. W. (1989) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.86.10.3699 · Cited by 261 · see citing works
Atkinson, N. J., Witteveldt, J., Evans, D. J., & Simmonds, P. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku075 · Cited by 192 · see citing works
Self-citationBeutler, E., Gelbart, T., Han, J. H., Koziol, J. A., & Beutler, B. (1989) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.86.1.192 · Cited by 189 · see citing works
Preferred and avoided codon pairs in three domains of life
Tats, A., Tenson, T., & Remm, M. (2008) · BMC Genomics · doi:10.1186/1471-2164-9-463 · Cited by 112 · cited 3× · see citing works
“…consistent underand over-representations of codon pairs (CPs) in all organisms (Yarus and Folley, 1985; Gutman and Hatfield, 1989; Boycheva et al., 2003; Moura et al., 2005; Tats et al., 2008) that have been proposed to influence gene expression through alterations in translation efficiency.”
BackgroundMoura, G., Pinheiro, M., Arrais, J., Gomes, A. C., Carreto, L., Freitas, A., Oliveira, J. L., & Santos, M. A. S. (2007) · PLoS ONE · doi:10.1371/journal.pone.0000847 · Cited by 107 · see citing works
Yang, C., Skiena, S., Futcher, B., Mueller, S., & Wimmer, E. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1307473110 · Cited by 103 · see citing works
Comparative context analysis of codon pairs on an ORFeome scale
Moura, G., Pinheiro, M., Silva, R., Miranda, I., Afreixo, V., Dias, G., Freitas, A., Oliveira, J. L., & Santos, M. A. (2005) · Genome Biology · doi:10.1186/gb-2005-6-3-r28 · Cited by 90 · see citing works
Martrus, G., Nevot, M., Andres, C., Clotet, B., & Martinez, M. A. (2013) · Retrovirology · doi:10.1186/1742-4690-10-78 · Cited by 85 · see citing works
Flagged first, then by citations
Research works printed but not linked: 30
- Bennetzen JL Hall BD 1982 Codon selection in yeast The Journal of Biological Chemistry 257 3026 3031 7037777
- Boycheva S Chkodrov G Ivanov I 2003 Codon pairs in the genome of Escherichia coli Bioinformatics 19 987 998 10.1093/bioinformatics/btg082 12761062 · DOI
- Buchan JR Aucott LS Stansfield I 2006 tRNA properties help shape codon pair preferences in open reading frames Nucleic Acids Research 34 1015 1027 10.1093/nar/gkj488 16473853 PMC1363775 · DOI · Paper page
- Burns CC Campagnoli R Shaw J Vincent A Jorba J Kew O 2009 Genetic inactivation of poliovirus infectivity by increasing the frequencies of CpG and UpA dinucleotides within and across synonymous capsid region codons Journal of Virology 83 9957 9969 10.1128/JVI.00508-09 19605476 PMC2747992 · DOI · Paper page
- Chevance FF Le Guyon S Hughes KT 2014 The effects of codon context on in vivo translation speed PLOS Genetics 10 e1004392 10.1371/journal.pgen.1004392 24901308 PMC4046918 · DOI
- Coleman JR Papamichail D Skiena S Futcher B Wimmer E Mueller S 2008 Virus attenuation by genome-scale changes in codon pair bias Science 320 1784 1787 10.1126/science.1155761 18583614 PMC2754401 · DOI · Paper page
- Duan J Antezana MA 2003 Mammalian mutation pressure, synonymous codon choice, and mRNA degradation Journal of Molecular Evolution 57 694 701 10.1007/s00239-003-2519-1 14745538 · DOI
- Everitt AR Clare S Pertel T John SP Wash RS Smith SE Chin CR Feeley EM Sims JS Adams DJ Wise HM Kane L Goulding D Digard P Anttila V Baillie JK Walsh TS Hume DA Palotie A Xue Y Colonna V Tyler-Smith C Dunning J Gordon SB Smyth RL Openshaw PJ Dougan G Brass AL Kellam P 2012 IFITM3 restricts the morbidity and mortality associated with influenza Nature 484 519 523 10.1038/nature10921 22446628 PMC3648786 · DOI
- Folley LS Yarus M 1989 Codon contexts from weakly expressed genes reduce expression in vivo Journal of Molecular Biology 209 359 378 10.1016/0022-2836(89)90003-X 2511323 · DOI
- Gingold H Pilpel Y 2011 Determinants of translation efficiency and accuracy Molecular Systems Biology 7 481 10.1038/msb.2011.14 21487400 PMC3101949 · DOI
- Hambleton S Goodbourn S Young DF Dickinson P Mohamad SM Valappil M McGovern N Cant AJ Hackett SJ Ghazal P Morgan NV Randall RE 2013 STAT2 deficiency and susceptibility to viral illness in humans Proceedings of the National Academy of Sciences of USA 110 3053 3058 10.1073/pnas.1220098110 PMC3581986 23391734 · DOI
- Irwin B Heck JD Hatfield GW 1995 Codon pair utilization biases influence translational elongation step times The Journal of Biological Chemistry 270 22801 22806 10.1074/jbc.270.39.22801 7559409 · DOI · Paper page
- Karlin S Doerfler W Cardon LR 1994 Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? Journal of Virology 68 2889 2897 8151759 PMC236777
- Le Nouen C Brock LG Luongo C McCarty T Yang L Mehedi M Wimmer E Mueller S Collins PL Buchholz UJ DiNapoli JM 2014 Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization Proceedings of the National Academy of Sciences of USA 111 13169 13174 10.1073/pnas.1411290111 PMC4246931 25157129 · DOI
- Mueller S Coleman JR Papamichail D Ward CB Nimnual A Futcher B Skiena S Wimmer E 2010 Live attenuated influenza virus vaccines by computer-aided rational design Nature Biotechnology 28 723 726 10.1038/nbt.1636 PMC2902615 20543832 · DOI · Paper page
- Ni YY Zhao Z Opriessnig T Subramaniam S Zhou L Cao D Cao Q Yang H Meng XJ 2014 Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus Virology 450-451 132 139 10.1016/j.virol.2013.12.009 24503075 · DOI
- Pothlichet J Quintana-Murci L 2013 The genetics of innate immunity sensors and human disease International Reviews of Immunology 32 157 208 10.3109/08830185.2013.777064 23570315 · DOI
- Puigbò P Bravo IG Garcia-Vallve S 2008 CAIcal: a combined set of tools to assess codon usage adaptation Biology Direct 3 38 10.1186/1745-6150-3-38 18796141 PMC2553769 · DOI
- Ramsey PH 1989 Critical values for spearman's rank order correlation Journal of Educational Statistics 14 245 253 10.2307/1165017 · DOI
- Rima BK McFerran NV 1997 Dinucleotide and stop codon frequencies in single-stranded RNA viruses The Journal of General Virology 78 2859 2870 9367373
- Sharp PM Bailes E Grocock RJ Peden JF Sockett RE 2005 Variation in the strength of selected codon usage bias among bacteria Nucleic Acids Research 33 1141 1153 10.1093/nar/gki242 15728743 PMC549432 · DOI
- Simmen MW 2008 Genome-scale relationships between cytosine methylation and dinucleotide abundances in animals Genomics 92 33 40 10.1016/j.ygeno.2008.03.009 18485662 · DOI
- Simmonds P 2012 SSE: a nucleotide and amino acid sequence analysis platform BMC Research Notes 5 50 10.1186/1756-0500-5-50 22264264 PMC3292810 · DOI
- Simmonds P Xia W Baillie JK McKinnon K 2013 Modelling mutational and selection pressures on dinucleotides in eukaryotic phyla –selection against CpG and UpA in cytoplasmically expressed RNA and in RNA viruses BMC Genomics 14 610 10.1186/1471-2164-14-610 24020411 PMC3829696 · DOI
- Thomas DL Thio CL Martin MP Qi Y Ge D O'Huigin C Kidd J Kidd K Khakoo SI Alexander G Goedert JJ Kirk GD Donfield SM Rosen HR Tobler LH Busch MP McHutchison JG Goldstein DB Carrington M 2009 Genetic variation in IL28B and spontaneous clearance of hepatitis C virus Nature 461 798 801 10.1038/nature08463 19759533 PMC3172006 · DOI
- Wang FP Li H 2009 Codon-pair usage and genome evolution Gene 433 8 15 10.1016/j.gene.2008.12.016 19159666 · DOI
- Wimmer E Mueller S Tumpey TM Taubenberger JK 2009 Synthetic viruses: a new opportunity to understand and prevent viral disease Nature Biotechnology 27 1163 1172 10.1038/nbt.1593 PMC2819212 20010599 · DOI
- Wright F 1990 The 'effective number of codons' used in a gene Gene 87 23 29 10.1016/0378-1119(90)90491-9 2110097 · DOI
- Wu X Wu S Li D Zhang J Hou L Ma J Liu W Ren D Zhu Y He F 2010 Computational identification of rare codons of Escherichia coli based on codon pairs preference BMC Bioinformatics 11 61 10.1186/1471-2105-11-61 20109184 PMC2828438 · DOI
- Yarus M Folley LS 1985 Sense codons are found in specific contexts Journal of Molecular Biology 182 529 540 10.1016/0022-2836(85)90239-6 3892014 · DOI
Cited by
Sorted by citations
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
Blitvich, B., & Firth, A. (2015) · Viruses · doi:10.3390/v7041927 · Cited by 323 · see citing works
Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias
Kunec, D., & Osterrieder, N. (2016) · Cell Reports · doi:10.1016/j.celrep.2015.12.011 · Cited by 159 · see citing works
“A study that analyzed a library of echovirus mutants - in which either CpG and TpA frequency or the overall CPB was kept constant while the other feature was altered - demonstrated that the increase of CpG and TpA frequencies impaired virus fitness but that alternation of CPB without changing the CpG and TpA frequency…”
BackgroundInfluentialThe Evolutionary Pathway to Virulence of an RNA Virus
Stern, A., Yeh, M. T., Zinger, T., Smith, M., Wright, C., Ling, G., Nielsen, R., Macadam, A., & Andino, R. (2017) · Cell · doi:10.1016/j.cell.2017.03.013 · Cited by 127 · see citing works
Ficarelli, M., Wilson, H., Pedro Galão, R., Mazzon, M., Antzin-Anduetza, I., Marsh, M., Neil, S. J., & Swanson, C. M. (2019) · eLife · doi:10.7554/elife.46767 · Cited by 135 · 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
New Vaccine Design and Delivery Technologies
Kanekiyo, M., Ellis, D., & King, N. P. (2019) · The Journal of Infectious Diseases · doi:10.1093/infdis/jiy745 · Cited by 69 · see citing works
Sequence-Specific Sensing of Nucleic Acids
Vabret, N., Bhardwaj, N., & Greenbaum, B. D. (2017) · Trends in Immunology · doi:10.1016/j.it.2016.10.006 · Cited by 45 · see citing works
Reply to Simmonds et al.: Codon pair and dinucleotide bias have not been functionally distinguished
Futcher, B., Gorbatsevych, O., Shen, S. H., Stauft, C. B., Song, Y., Wang, B., Leatherwood, J., Gardin, J., Yurovsky, A., Mueller, S., et al. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1507710112 · Cited by 22 · see citing works
- Authors
- Tulloch, F., Atkinson, N. J., Evans, D. J., Ryan, M. D., & Simmonds, P.
- Affiliations
- University of St Andrews (GB)
- Roslin Institute (GB)
- University of Warwick (GB)
- Infection and Immunity Division, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2014-12-09
- Volume
- 3
- Pages
- e04531-e04531
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC4383024
- OpenAlex
- W2163194377
- Cited by
- 178
- References
- 8
- Citations by year
- 2026: 12 · 2025: 16 · 2024: 2 · 2023: 13 · 2022: 8 · 2021: 17 · 2020: 24 · 2019: 21 · 2018: 15 · 2017: 22 · 2016: 13 · 2015: 15
- FWCI
- 6.84
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- St Andrews Research Repository (St Andrews Research Repository) (submittedVersion, cc-by)
- Funders
- Wellcome Trust (WT087628MA)
- Biotechnology and Biological Sciences Research Council (BB/K003801/1, BB/L004526/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
- Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
- Keywords
- echovirus 7, picornavirus, translation efficiency, live attenuated virus vaccine, innate immune response, vaccine safety
- MeSH terms
- Codon, Humans, RNA, Viral, Protein Biosynthesis, Vaccines, Attenuated, Viral Vaccines, Virus Replication, Dinucleoside Phosphates, CpG Islands, Base Pairing, Enterovirus B, Human, Muscle Cells, Cell Line, Tumor
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
