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Cited by 85RNA and protein synthesis mechanismsInfluenza Virus Research StudiesEvolution and Genetic Dynamics

Mechanism of Virus Attenuation by Codon Pair Deoptimization

Open access
Groenke, Nicole, Trimpert, Jakob, Merz, Sophie, Conradie, Andelé M., Wyler, Emanuel, Zhang, Hongwei, Hazapis, Orsalia-Georgia, Rausch, Sebastian +3 more·2020·Cell Reports
DOI 10.1016/j.celrep.2020.107586
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Abstract

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.

AI snapshot

Written from the paper’s full text.

TL;DR

Codon pair deoptimization attenuates influenza A virus primarily through underrepresented codon pairs, not through increased CpG/UpA dinucleotides. Recoded NA genes produce unstable mRNA and are poorly translated, reducing NA protein and viral fitness.

Key findings

  • Codon pair–deoptimized viruses 27-Min, 46-Med, and 72-Min were attenuated, whereas CpG-high viruses 72L-Max and 72H-Max were not. In A549 cells, the codon pair–deoptimized viruses produced 1,000–10,000 times lower infectious virus output, and their plaques were 3–5 times smaller than 27-WT.
  • In mice, lung infectious virus and NP/NA RNA levels were 10–1,000 times lower for 27-Min, 46-Med, and 72-Min than for 27-WT; 72-Min showed no detectable infectious virus at 5 dpi. These attenuated viruses induced complete protection against challenge with 5 × 10^5 PFU of 27-WT, with no body weight loss and virus cleared by 10 days after challenge.
  • Mechanistically, RNA synthesis rates did not differ significantly, but codon pair–deoptimized transcripts decayed faster and had strongly reduced translation efficiency. For 46-Med and 27-Min, translation almost completely stopped after 760 nucleotides, yielding a truncated NA polypeptide containing only the first 250 N-terminal amino acids; 72-Min had the lowest translation efficiency.
  • CpG/UpA dinucleotides were not the main cause: 72L-Max and 72H-Max contain 72 CpG dinucleotides and high UpA but replicated like 27-WT and produced comparable or increased NA protein, while 27-Min has the same CpG count as 27-WT (27) but minimal CPS and is attenuated.

Methods

  • The authors designed recoded variants of the neuraminidase (NA) gene in mouse-adapted influenza A virus A/WSN/1933 (H1N1), recoding only the central 339 codons (1,017 nt) and leaving the first 61 and last 54 codons unchanged. Variants differed in CpG dinucleotide number and average codon pair score (CPS).
  • In vitro work used MDCK, A549, and HEK293T cells with multi-step growth kinetics, plaque assays, western blotting, qRT-PCR, BrU pulse-chase labeling, flavopiridol transcription inhibition, ribosome profiling, and mass spectrometry.
  • In vivo work used female 6-week-old BALB/c mice: seven groups of nine mice were infected intranasally with 5 × 10^4 PFU of parental or recoded viruses; a vaccination experiment immunized eight mice and challenged them on day 28 with 5 × 10^5 PFU of 27-WT. Lung viral titers, RNA copy numbers, histopathology, antibody titers, and IFN-γ responses were measured.

Limitations

  • The paper states that single-step growth kinetics on MDCK cells have only limited discriminatory power for revealing compromised fitness of codon pair–deoptimized viruses, and it calls for future research to determine the contribution of individual codon pairs to mRNA destabilization and translation inhibition.
  • Evident from the text: the mechanistic dissection focuses on one recoded gene (NA) in one IAV strain and one mouse model, so generalization to other genes, viruses, or hosts is not established. Some mechanistic experiments were performed in transiently transfected HEK293T cells rather than in infected cells.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2020.107586

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

24 of 31 cited sources checked · 13 supported · 2 flagged · accuracy not yet measured

  • Interpretationown data

    Reduction of protein production is not the sum of small negative effects exerted by individual underrepresented, non-optimal codon pairs.

    “The identification of a clear premature translation termination point in different recoded genes (Figure 5G; Figure S6) suggests that reduction of protein production is not the sum of small negative effects exerted by individual underrepresented, non-optimal codon pairs (Coleman et al., 2008).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Coleman et al., 2008 source

      The cited paper reports that underrepresented codon pairs reduce translation, but it does not attribute that reduction to a clear premature termination point. Its discussion of PV-Min attenuation instead says that many mutations are consistent with causing “small, additive defects”—the opposite of the sentence’s proposed account. The latter finding concerns attenuation rather than a direct measurement of each pair’s effect on protein production. (LLM, conf=0.83)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    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).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Kunec and Osterrieder, 2016 source
    • cites Tulloch et al., 2014 source
  • Premisecitation

    Codon pair deoptimization (CPD) is a highly efficient virus attenuation strategy that utilizes suboptimal codon pairs to achieve attenuation of recoded viruses.

    “Codon pair deoptimization (CPD) is a highly efficient virus attenuation strategy that utilizes suboptimal codon pairs to achieve attenuation of recoded viruses.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Coleman et al., 2008 source
    • cites Mueller et al., 2010 source
  • Interpretationcitation

    The level of attenuation achieved by the elevation of CpG/UpA dinucleotide frequencies appears to be much weaker than that by CPD.

    “Although these studies are not directly comparable, the level of attenuation achieved by the elevation of CpG/UpA dinucleotide frequencies (Gaunt et al., 2016) appears to be much weaker than that by CPD (Yang et al., 2013).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Gaunt et al., 2016 source
    • cites Yang et al., 2013 source
  • Premisecitation

    MDCK cells produce only a relatively thin layer of mucus.

    “MDCK cells only produce a relatively thin layer of mucus (Barnard et al., 2019;Harush-Frenkel et al., 2008).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Barnard et al., 2019 source

      The Results section identifies A549 cells as secretory and reports their mucin secretion, but does not describe mucus production by MDCK cells. Searches for MDCK mucus, mucin, and a thin mucus layer found no passage establishing the claim in our copy; page numbers were not available. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites Harush-Frenkel et al., 2008 source
  • Classified as probably not a substantial claim. 29
  • May combine two claims. 45

  • Classified as probably not a substantial claim.

    Premisecitation

    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).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Broadbent et al., 2016 source
    • cites Coleman et al., 2008 source

      In the passage describing the translation assay (section labeled Abstract; no page given), Coleman et al. report that underrepresented codon pairs reduced translation and say this is probably sufficient to explain the attenuated phenotype. They leave open why particular codon pairs are underrepresented, but do not present the mechanism of attenuation as wholly unknown. (LLM, conf=0.85)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Futcher et al., 2015 source

  • May combine two claims.

    Premisecitation

    CPD can reduce stability of recoded mRNA and induce temperature-sensitivity in mutant viruses.

    “In addition, it was shown that CPD can reduce stability of recoded mRNA (Broadbent et al., 2016;Yang et al., 2013) and induce temperature-sensitivity in mutant viruses (Le Noue ¨n et al., 2014;Yang et al., 2013).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Broadbent et al., 2016 source

      The Results report reduced HA mRNA levels in cells infected with the recoded virus. The Discussion says this might reflect either decreased transcription or increased mRNA degradation, so the paper does not establish reduced mRNA stability. The temperature-sensitivity claim is cited to other papers, not Broadbent et al. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Yang et al., 2013 source
    • cites Le Noue ¨n et al., 2014

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    The prevailing hypothesis is that the large number of underrepresented codon pairs interferes with efficient protein production or processing.

    “While different mechanisms have been proposed, the prevailing hypothesis is that the large number of underrepresented codon pairs interferes with efficient protein production or processing (Coleman et al., 2008;Shen et al., 2015).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Coleman et al., 2008 source

      The paper reports that hundreds of underrepresented codon pairs reduced protein translation in recoded poliovirus, supporting the protein-production mechanism (Abstract; page not supplied). It does not establish that this is the prevailing hypothesis across settings or address impaired protein processing. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Shen et al., 2015 source

      The Discussion says that poor codon pairs might affect polyprotein synthesis or processing, which supports the proposed mechanism. It also says the precise mechanism remains unknown and lists other possibilities; it does not identify impaired production or processing as the prevailing hypothesis. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing). May combine two claims.

    Premisecitation

    An alternate hypothesis proposes that CpG dinucleotides are the sole cause of attenuation because they act as triggers of antiviral immune responses.

    “Thus, an alternate hypothesis proposes that CpG dinucleotides are the sole cause of attenuation because they act as triggers of antiviral immune responses (Greenbaum et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Greenbaum et al., 2009 source

      The Discussion (page unavailable) proposes that CpG-containing RNA motifs can trigger an innate immune response. But the paper does not say CpG dinucleotides are the sole cause of attenuation; its Results specify that CpGs in an A/U context, rather than all CpGs, are preferentially selected against. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    CPD rearranges the positions of existing synonymous codons in a viral gene, without changing the codon bias or amino acid composition of the encoded protein.

    “CPD rearranges the positions of existing synonymous codons in a viral gene, without changing the codon bias or amino acid composition of the encoded protein (Coleman et al., 2008;Mueller et al., 2010).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Coleman et al., 2008 source
    • cites Mueller et al., 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The host zinc-finger antiviral protein (ZAP) binds specifically to CpG-rich RNAs and targets them for degradation by the RNA exosome.

    “Recent studies showed that the host zinc-finger antiviral protein (ZAP) is a long-suspected single-stranded RNA (ssRNA) CpG receptor because it binds specifically to CpG-rich RNAs and targets them for degradation by the RNA exosome (Ficarelli et al., 2019;Takata et al., 2017).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Ficarelli et al., 2019 source

      The Introduction states that ZAP binds CpG-rich regions of HIV-1 RNA and targets them for degradation. The Discussion reports an interaction with the exosome, but says knockdown of several degradation-pathway proteins did not substantially rescue virus production; it presents exosome involvement in this setting as a possible mechanism, not an established route of degradation. No page numbers are available in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Takata et al., 2017 source

      The paper reports that ZAP binds directly and selectively to CG-containing RNA and causes cytoplasmic depletion of high-CG RNA (Abstract section; page not provided). It mentions RNA exosome pathways as targets in a screening experiment, but does not establish that the exosome degrades the RNAs bound by ZAP. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Human alveolar carcinoma A549 cells are secretory cells and produce substantial amounts of mucin.

    “In contrast, human alveolar carcinoma A549 cells are secretory cells and produce substantial amounts of mucin (Barnard et al., 2019;Berger et al., 1999).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Barnard et al., 2019 source
    • cites Berger et al., 1999 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Codon pair bias can be quantified using the codon pair score (CPS) statistics, where statistically underrepresented codon pairs have negative and overrepresented codon pairs have positive CPS values.

    “Codon pair bias can be quantified using the codon pair score (CPS) statistics (Coleman et al., 2008), where statistically underrepresented codon pairs have negative and overrepresented codon pairs have positive CPS values.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Coleman et al., 2008 source

      The passage labeled Abstract says codon pairs can be under- or overrepresented relative to statistical expectations, and describes a codon pair bias (CPB) score for a recoded virus. It does not define a codon pair score (CPS) or state that negative CPS values indicate underrepresentation and positive values indicate overrepresentation. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CPD of vertebrate viruses also increases the frequency of TpA/UpA dinucleotides in recoded sequences, albeit to a lesser degree than CpG.

    “In addition to CpG dinucleotides, CPD of vertebrate viruses also increases the frequency of TpA/UpA dinucleotides in recoded sequences, albeit to a lesser degree (Kunec and Osterrieder, 2016).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kunec and Osterrieder, 2016 source

  • Classified as probably not a substantial claim.

    Premisecitation

    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).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Atkinson et al., 2014 source
    • cites Burns et al., 2009 source
    • cites Tulloch et al., 2014 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    IAV double mutants with codon pair deoptimized NA and hemagglutinin genes were completely attenuated in BALB/c mice and ferrets.

    “In a similar study, IAV double mutants with codon pair deoptimized NA and hemagglutinin genes were completely attenuated in BALB/c mice (Yang et al., 2013) and ferrets (Broadbent et al., 2016).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Yang et al., 2013 source
    • cites Broadbent et al., 2016 source

      The Broadbent citation is attached to the ferret finding, not the BALB/c mouse finding. The Discussion reports that the HA-and-NA-deoptimized virus was clinically attenuated in ferrets, but also says its replication was not restricted and that further de-optimization could increase attenuation. It therefore does not support calling the virus completely attenuated. (LLM, conf=0.91)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The 72-Min virus showed strongest attenuation and no infectious virus could be detected in lungs of infected animals at 5 dpi.

    “The 72-Min virus showed strongest attenuation and no infectious virus could be detected in lungs of infected animals at 5 dpi.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The number of infectious viruses as well as NP and NA RNA copy numbers were 10-1,000 times lower in lungs of mice infected with the codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min when compared to the 27-WT control group.

    “On the other hand, the number of infectious viruses (Figure 3B) as well as NP (Figure 3C) and NA RNA copy numbers (Figure S2A) were 10-1,000 times lower in lungs of mice infected with the codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min when compared to the 27-WT control group.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 72-Min gene had the lowest translation efficiency, which is in good agreement with the strong attenuation of the corresponding mutant in vitro and in vivo.

    “The 72-Min gene had the lowest translation efficiency, which is in good agreement with the strong attenuation of the corresponding mutant in vitro and in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Almost all mice infected with the mutant 72H-Max virus, and most mice infected with the mutant 72L-Max virus, developed severe pneumonia within the first few days after infection.

    “Similarly, almost all mice infected with the mutant 72H-Max virus, and most mice infected with the mutant 72L-Max virus, developed severe pneumonia within the first few days after infection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    While 46-Med and 27-Min also exhibited reduced translation efficiency, we also identified an almost complete stop of translation after 760 nucleotides.

    “While 46-Med and 27-Min also exhibited reduced translation efficiency, we also identified an almost complete stop of translation after 760 nucleotides (Figure 5G; Figure S6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was no detectable necrosis and only mild inflammation in the lungs of protected animals.

    “There was no detectable necrosis (Figure 4C; Figure S4) and only mild inflammation (Figures 4D and 4F; Figure S4) in the lungs of protected animals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 72-Min virus was virtually apathogenic and lungs of mice infected with the 72-Min virus resembled those of the control group, since no histopathological signs of lung disease were present.

    “The 72-Min virus was virtually apathogenic and lungs of mice infected with the 72-Min virus resembled those of the control group, since no histopathological signs of lung disease were present.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The degradation rates of mRNA NA transcripts were almost identical to those obtained in the BrU chase experiment, confirming that mRNA species made by codon pair-deoptimized genes are less stable than those produced by the parental 27-WT gene.

    “The degradation rates of mRNA NA transcripts were almost identical to those obtained in the BrU chase experiment (Figure 5D), confirming that mRNA species made by codon pair-deoptimized genes are less stable than those produced by the parental 27-WT gene.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Suboptimal codon pairs reduce mRNA stability and throttle or abrogate translation.

    “Suboptimal codon pairs reduce mRNA stability and throttle or abrogate translation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The RNA levels produced by the 27-WT or 72H-Max, 72L-Max genes remained relatively stable up to 2 h after labeling; however, the RNA produced by the codon pair-deoptimized genes 72-Min, 46-Med, and 27-Min was unstable and was rapidly degraded.

    “The RNA levels produced by the 27-WT or 72H-Max, 72L-Max genes remained relatively stable up to 2 h after labeling; however, the RNA produced by the codon pair-deoptimized genes 72-Min, 46-Med, and 27-Min was unstable and was rapidly degraded (Figure 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The NA protein levels correlated very well with the levels produced by the viruses in infected cells, confirming that the capacity of recoded genes to produce proteins is an inherent characteristic of the sequence, and independent of virus replication.

    “It is important to note that the NA protein levels correlated very well with the levels produced by the viruses in infected cells (Figure 2F), confirming that the capacity of recoded genes to produce proteins is an inherent characteristic of the sequence, and independent of virus replication.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice infected with the codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min and the single surviving mouse infected with 72L-Max showed high antibody titers, indicating that these viruses induced robust immune responses.

    “Mice infected with the codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min and the single surviving mouse infected with 72L-Max showed high antibody titers (Figures S2B and S2C), indicating that these viruses induced robust immune responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NA mRNA levels were only moderately decreased in the case of 72L-Max (75% of the 27-WT) and increased in the case of 72H-Max (170% of the 27-WT).

    “In contrast, NA mRNA levels were only moderately decreased in the case of 72L-Max (75% of the 27-WT) and increased in the case of 72H-Max (170% of the 27-WT).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The codon pair-deoptimized NA genes 27-Min, 46-Med, and 72-Min produced less NA protein, while genes with increased number of CpG dinucleotides produced comparable (72L-Max) or increased (72H-Max) amounts of NA protein than the 27-WT gene.

    “The codon pair-deoptimized NA genes 27-Min, 46-Med, and 72-Min produced less NA protein, while genes with increased number of CpG dinucleotides produced comparable (72L-Max) or increased (72H-Max) amounts of NA protein than the 27-WT gene (Figure 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All three attenuated viruses induced strong adaptive immune responses, detected as high anti-IAV antibody titers and prominent virus-specific interferon-gamma (IFN-g) responses in immunized mice.

    “All three attenuated viruses induced strong adaptive immune responses, detected as high anti-IAV antibody titers (Figure 4B) and prominent virus-specific interferon-gamma (IFN-g) responses in immunized mice (Figure S4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The parental 27-WT, 72L-Max, and 72H-Max viruses replicated very efficiently and to comparable levels in the lungs of infected mice.

    “The parental 27-WT, 72L-Max, and 72H-Max viruses replicated very efficiently and to comparable levels in the lungs of infected mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In MDCK cells, only the codon pair-deoptimized viruses 46-Med and 72-Min replicated less efficiently than the 27-WT virus, and their viral titers were typically 10 times lower than those of the 27-WT virus.

    “In MDCK cells, only the codon pair-deoptimized viruses 46-Med and 72-Min replicated less efficiently than the 27-WT virus, and their viral titers Article were typically $10 times lower than those of the 27-WT virus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In A549 cells, three codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min replicated with severely reduced kinetics when compared to the 27-WT virus, and we recorded an $1,000-10,000 times lower output of infectious virus particles by the codon pair-deoptimized viruses.

    “In contrast, three codon pair-deoptimized viruses 27-Min, 46-Med, and 72-Min replicated with severely reduced kinetics when compared to the 27-WT virus, and we recorded an $1,000-10,000 times lower output of infectious virus particles by the codon pair-deoptimized viruses (Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In transiently transfected cells, RNA transcripts produced by codon pair-deoptimized genes have reduced stability and translatability.

    “The analysis of RNA kinetics in transiently transfected cells showed that the stability and translatability of RNA transcripts produced by codon pair-deoptimized genes is reduced (Figures 5D-5F).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    46-Med has 46 CpG dinucleotides.

    “46-Med | reduced CPS | 46 | 77 | À0.23 | 0.74 | 61.4 | 83.6”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The increase of CpG dinucleotides in codon pair-deoptimized sequences had negligible effects on protein production and virus attenuation.

    “While the increase of CpG (and UpA) dinucleotides in recoded sequences might contribute to virus attenuation, our data show that the increase of CpG dinucleotides in codon pair-deoptimized sequences had negligible effects on protein production and virus attenuation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    27-Min has 27 CpG dinucleotides.

    “27-Min | same NNC-GNN as 27-WT, minimal CPS | 27 | 82 | À0.22 | 0.74 | 52.5 | 80.2”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Mock control group shows high titers after 27-WT challenge.

    “Mock control group shows high titers after 27-WT challenge.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    72-Min has 100 UpA dinucleotides.

    “72-Min | minimal CPS | 72 | 100 | À0.45 | 0.74 | 37.8 | 75.6”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    72-Min has 72 CpG dinucleotides.

    “72-Min | minimal CPS | 72 | 100 | À0.45 | 0.74 | 37.8 | 75.6”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The 72L-Max mutant contains a high number of UpA and CpG dinucleotides but did not show any attenuation of replication.

    “For example, the 72L-Max mutant contains a high number of UpA and CpG dinucleotides but did not show any attenuation of replication (Figure 2; Table 1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    72-Min has a minimal codon pair score (CPS) of -0.45.

    “72-Min | minimal CPS | 72 | 100 | À0.45 | 0.74 | 37.8 | 75.6”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    27-Min has 82 UpA dinucleotides.

    “27-Min | same NNC-GNN as 27-WT, minimal CPS | 27 | 82 | À0.22 | 0.74 | 52.5 | 80.2”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Two mice from the control group survived the challenge infection and started to gain weight on day 4 after challenge.

    “Two mice from the control group survived the challenge infection and started to gain weight on day 4 after challenge.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Replacing non-optimal codon pairs with optimal codon pairs in 72-Min restored protein production and reversed its attenuated phenotype (producing 72L-Max and 72H-Max).

    “Similarly, we restored protein production and reversed the attenuated phenotype of the 72-Min mutant virus by replacing the non-optimal codon pairs with optimal codon pairs, and thereby producing the 72L-Max and 72H-Max genes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    27-Min has a minimal codon pair score (CPS) of -0.22.

    “27-Min | same NNC-GNN as 27-WT, minimal CPS | 27 | 82 | À0.22 | 0.74 | 52.5 | 80.2”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Neither CpG dinucleotides nor codon pairs of the NNC-GNN type play an important role in the attenuation of recoded viruses 72L-Max and 72H-Max.

    “We showed that neither CpG dinucleotides nor codon pairs of the NNC-GNN type play an important role in the attenuation of recoded viruses (72L-Max and 72H-Max).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    46-Med has a reduced codon pair score (CPS) of -0.23.

    “46-Med | reduced CPS | 46 | 77 | À0.23 | 0.74 | 61.4 | 83.6”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Viral NA RNA stability was slightly reduced in 46-Med and markedly reduced in 72-Min and 27-Min viruses.

    “The stability of viral NA RNA was slightly reduced in 46-Med, and markedly reduced in 72-Min and 27-Min viruses (Figure 6B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The low average CPS of 72L-Max is solely due to NNC-GNN codon pairs, but the virus mutant 72L-Max is not attenuated.

    “For example, the low average CPS of 72L-Max is solely due to NNC-GNN codon pairs (Figures 1B and 1C), but the virus mutant 72L-Max is not attenuated.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    46-Med has 77 UpA dinucleotides.

    “46-Med | reduced CPS | 46 | 77 | À0.23 | 0.74 | 61.4 | 83.6”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Introduction of underrepresented codon pairs into recoded NA genes severely impaired the stability of mRNA transcripts and their translation efficiency.

    “Introduction of underrepresented codon pairs into recoded NA genes did not have a measurable effect on RNA transcription (Figure 5C), but it severely impaired the stability of mRNA transcripts and their translation efficiency (Figures 5D-5F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Parental and all mutant viruses replicated with similar kinetics and produced similar titers on MDCK cells.

    “We observed that the parental and all mutant viruses replicated with similar kinetics and produced similar titers on MDCK cells (Figure 2A), but three codon pair-deoptimized viruses-27-Min, 46-Med, and 72-Min-showed a strongly attenuated phenotype on A549 cells (Figure 2B), growing to 100-1,000 times lower titer than the parental virus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Decreased mRNA stability and reduced translation efficiency underlie the attenuation by CPD.

    “We show that decreased mRNA stability and reduced translation efficiency underlie the attenuation by CPD.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Underrepresented codon pairs were solely responsible for attenuation of recoded viruses, whereas CpG dinucleotide frequencies had a negligible effect.

    “We show that underrepresented codon pairs were solely responsible for attenuation of recoded viruses, whereas CpG dinucleotide frequencies had a negligible effect.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Underrepresented codon pairs are the cause of reduced translation efficiency and lead to reduced protein production.

    “This corresponded well with the results showing that underrepresented codon pairs are the cause of reduced translation efficiency and lead to reduced protein production.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The identity of codon pairs is another critical determinant of mRNA stability, which profoundly affects protein output and, ultimately, fitness of recoded viruses.

    “Here we demonstrate that the identity of codon pairs is another critical determinant of mRNA stability, which profoundly affects protein output and, ultimately, fitness of recoded viruses.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Lung sections were evaluated by microscopy in a blinded fashion.

    “Lung sections stained with hematoxylin and eosin were evaluated by microscopy in a blinded fashion (Dietert et al., 2017).”
    • Also stated in: § Additional information · plain
      “Lung sections were evaluated by microscopy in a blinded fashion.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mice were randomly distributed to groups.

    “Female 6-week-old BALB/c mice were purchased from Janvier, distributed randomly to 7 groups of 9 animals, and infected intranasally with 5 3 10 4 PFU of IAV in 30 ml DMEM medium under ketamine and xylazine anesthesia (Plumb, 2015).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Neither UpA nor CpG dinucleotides play a prominent role in the attenuation of recoded IAV mutants.

    “Thus, we conclude that neither UpA nor CpG dinucleotides play a prominent role in the attenuation of recoded IAV mutants.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Only sequencing reads with mapping quality 24 or higher were kept, which also filtered out reads mapping to the constant regions at the beginning and end of the NA constructs.

    “Only reads with mapping quality 24 or higher were kept, which also filtered out reads mapping to the constant regions at the beginning and end of the NA constructs.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Incubation with a purified goat antibody at the same dilution instead of primary antibody served as a negative control.

    “Incubation with a purified goat antibody at the same dilution instead of primary antibody served as a negative control.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Sequences of recoded NA genes were confirmed by Sanger sequencing.

    “To confirm sequences of recoded NA genes, viral RNA was isolated using RTP DNA/RNA Virus Mini Kit (Stratec SE), reverse transcribed with M-MLV reverse transcriptase (Promega) and sequenced by Sanger sequencing.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Beta-actin was used as loading control in western blot analyses.

    “Beta-actin was used as loading control.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The western blot images are representative of three independent biological experiments.

    “The images are representative of three independent biological experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The unique design of the recoded NA gene variants enabled comparison of mutant viruses that either had the same number of CpG dinucleotides, or nearly identical CPS, but all contained exactly the same codons.

    “The unique design of our recoded NA gene variants enabled us to compare biological properties of mutant viruses that either had the same number of CpG dinucleotides, or nearly identical CPS (Figure 1B), but all contained exactly the same codons.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisereasoning

    CPD of vertebrate virus genomes leads to an accumulation of NNC-GNN codon pairs in recoded sequences because they have extremely low CPS.

    “Since codon pairs of the NNC-GNN type have extremely low CPS, CPD of vertebrate virus genomes leads to an accumulation of these codon pairs in recoded sequences.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The principal role in attenuation by CPD is exerted by underrepresented codon pairs, which reduce the stability of recoded mRNA and inhibit efficient translation.

    “We argue that the principal role in attenuation by CPD is exerted by underrepresented codon pairs, which reduce the stability of recoded mRNA and inhibit efficient translation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductreasoning

    Only the central part of the NA ORF was recoded, leaving terminal regions unmodified, to ensure unaltered packaging and replication of the recoded NA segments.

    “To ensure unaltered packaging and replication of the recoded NA segments, only the central part of the NA ORF (1,017 nt, 339 codons) was recoded, leaving the first 183 nt (61 codons) and the last 162 nt (54 codons) of the ORF unmodified.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Plaque diameters were determined from 100 randomly selected plaques of each virus.

    “The plaque diameters of 100 randomly selected plaques of each virus were determined with ImageJ software (Schneider et al., 2012).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    As a control protein, supernatant of uninfected MDCK.2 cells was used.

    “As a control protein supernatant of uninfected MDCK.2 cells was used.”
    Quote in the paperNot a substantial claim
67 of 67

  • The paper relies on this without support.

    The mouse-adapted IAV strain A/WSN/1933 is an appropriate model for studying influenza virus attenuation and pathogenesis.

    “As an experimental model we used the mouse-adapted IAV strain A/WSN/1933 (H1N1), which causes acute respiratory disease in immunocompetent mice (Stuart-Harris, 1939).”

  • The paper relies on this without support.

    The NA gene is representative of other recoded viral genes for the mechanism of CPD attenuation.

    “We chose the NA gene because it is amenable to recoding and does not have alternative reading frames (Mueller et al., 2010;Yang et al., 2013).”

  • The paper relies on this without support.

    A549 cells harbor a layer of mucus that is rich in strongly sialylated glycoproteins.

    “In addition, A549 cells, like epithelial cells of the respiratory tract, harbor a layer of mucus that is rich in strongly sialylated glycoproteins.”
3 of 3

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • MDCK growth is described as both reduced and similar

    Minor

    The Results say 46-Med and 72-Min typically reached titers about 10 times lower than 27-WT in MDCK cells. The Discussion instead says all mutant viruses produced similar titers to the parental virus in those cells.

  • Faster RNA decay is attributed to the opposite virus group

    Minor

    One sentence attributes faster NA RNA decay to codon pair-optimized viruses. The next identifies the viruses with reduced RNA stability as 46-Med, 72-Min, and 27-Min, which the paper classifies as codon pair-deoptimized.

2 of 2

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Authors
Groenke, N., Trimpert, J., Merz, S., Conradie, A. M., Wyler, E., Zhang, H., Hazapis, O.-G., Rausch, S., Landthaler, M., Osterrieder, N., & Kunec, D.
Affiliations
  • Freie Universität Berlin (DE)
  • Max Delbrück Center (DE)
Corresponding author
Osterrieder, Nikolaus
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2020-04
Volume
31
Issue
4
Pages
107586
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2020.107586
OpenAlex
W3019259734
Semantic Scholar
da526af25d700ed48bf10e44c91f9e4f045b5db5
Cited by
85
References
49
Citations by year
2026: 9 · 2025: 14 · 2024: 7 · 2023: 20 · 2022: 12 · 2021: 19 · 2020: 4
FWCI
3.80
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Deutsche Forschungsgemeinschaft (143/12-1, OS 143/9-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Health Sciences › Medicine › Epidemiology › Influenza Virus Research Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
Keywords
viral attenuation, influenza A virus, codon pair bias, mRNA stability, translation efficiency, CpG, protein production, synonymous codons
MeSH terms
Animals, Codon, DNA Viruses, Humans, Viral Proteins, Genome, Viral, Mice

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.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

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Statistics and arithmetic are recomputed, not judged.

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