Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Attenuation of RNA viruses by redirecting their evolution in sequence space

Nature Microbiology volume 2, Article number: 17088 (2017) Cite this article

Subjects

Abstract

RNA viruses pose serious threats to human health. Their success relies on their capacity to generate genetic variability and, consequently, on their adaptive potential. We describe a strategy to attenuate RNA viruses by altering their evolutionary potential. We rationally altered the genomes of Coxsackie B3 and influenza A viruses to redirect their evolutionary trajectories towards detrimental regions in sequence space. Specifically, viral genomes were engineered to harbour more serine and leucine codons with nonsense mutation targets: codons that could generate Stop mutations after a single nucleotide substitution. Indeed, these viruses generated more Stop mutations both in vitro and in vivo, accompanied by significant losses in viral fitness. In vivo, the viruses were attenuated, generated high levels of neutralizing antibodies and protected against lethal challenge. Our study demonstrates that cornering viruses in ‘risky’ areas of sequence space may be implemented as a broad-spectrum vaccine strategy against RNA viruses.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Construction of 1-to-Stop and NoStop RNA viruses.
Figure 2: The 1-to-Stop CVB3 virus is highly sensitive to mutation.
Figure 3: The 1-to-Stop influenza A viruses are highly sensitive to mutation.
Figure 4: The 1-to-Stop viruses are attenuated in vivo.
Figure 5: Attenuation of ‘SpeedyStop’ virus, 1-to-Stop CVB3 coupled with a mutator polymerase.
Figure 6: Re-localizing viruses to an inhospitable area of the fitness landscape.

Similar content being viewed by others

References

  1. Domingo, E. & Holland, J. J. RNA virus mutations and fitness for survival. Annu. Rev. Microbiol. 51, 151–178 (1997).

    Article  CAS  Google Scholar 

  2. Bordería, A. V. et al. Group selection and contribution of minority variants during virus adaptation determines virus fitness and phenotype. PLoS Pathog. 11, e1004838 (2015).

    Article  Google Scholar 

  3. Acevedo, A., Brodsky, L. & Andino, R. Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature 505, 686–690 (2014).

    Article  CAS  Google Scholar 

  4. Lauring, A. S., Acevedo, A., Cooper, S. B. & Andino, R. Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus. Cell Host Microbe 12, 623–632 (2012).

    Article  CAS  Google Scholar 

  5. Hall, A. R., Griffiths, V. F., MacLean, R. C. & Colegrave, N. Mutational neighbourhood and mutation supply rate constrain adaptation in Pseudomonas aeruginosa. Proc. Biol. Sci. 277, 643–650 (2010).

    Article  CAS  Google Scholar 

  6. Wilke, C. O. Adaptive evolution on neutral networks. Bull. Math. Biol. 63, 715–730 (2001).

    Article  CAS  Google Scholar 

  7. Wilke, C. O., Wang, J. L., Ofria, C., Lenski, R. E. & Adami, C. Evolution of digital organisms at high mutation rates leads to survival of the flattest. Nature 412, 331–333 (2001).

    Article  CAS  Google Scholar 

  8. Sanjuán, R. Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies. Philos. Trans. R. Soc. Lond. B 365, 1975–1982 (2010).

    Article  Google Scholar 

  9. Cuevas, J. M., Domingo-Calap, P. & Sanjuán, R. The fitness effects of synonymous mutations in DNA and RNA viruses. Mol. Biol. Evol. 29, 17–20 (2012).

    Article  CAS  Google Scholar 

  10. Sanjuán, R., Moya, A. & Elena, S. F. The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc. Natl Acad. Sci. USA 101, 8396–8401 (2004).

    Article  Google Scholar 

  11. Anderson, J. P., Daifuku, R. & Loeb, L. A. Viral error catastrophe by mutagenic nucleosides. Annu. Rev. Microbiol. 58, 183–205 (2004).

    Article  CAS  Google Scholar 

  12. Perales, C., Martín, V. & Domingo, E. Lethal mutagenesis of viruses. Curr. Opin. Virol. 1, 419–422 (2011).

    Article  CAS  Google Scholar 

  13. Elena, S. F. RNA virus genetic robustness: possible causes and some consequences. Curr. Opin. Virol. 2, 525–530 (2012).

    Article  CAS  Google Scholar 

  14. Coleman, J. R. et al. Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784–1787 (2008).

    Article  CAS  Google Scholar 

  15. Tulloch, F. et al. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife 3, e04531 (2015).

    Article  Google Scholar 

  16. Carrasco, P., Daròs, J. A., Agudelo-Romero, P. & Elena, S. F. A real-time RT–PCR assay for quantifying the fitness of tobacco etch virus in competition experiments. J. Virol. Methods 139, 181–188 (2007).

    Article  CAS  Google Scholar 

  17. Gnädig, N. F. et al. Coxsackievirus B3 mutator strains are attenuated in vivo. Proc. Natl Acad. Sci. USA 109, E2294–E2303 (2012).

    Article  Google Scholar 

  18. Levi, L. I. et al. Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds. PLoS Pathog. 6, e1001163 (2010).

    Article  Google Scholar 

  19. McDonald, S. et al. Design of a genetically stable high fidelity Coxsackievirus B3 polymerase that attenuates virus growth in vivo. J. Biol. Chem. 291, 13999–14011 (2016).

    Article  CAS  Google Scholar 

  20. Isakov, O. et al. Deep sequencing analysis of viral infection and evolution allows rapid and detailed characterization of viral mutant spectrum. Bioinformatics 31, 2141–2150 (2015).

    Article  CAS  Google Scholar 

  21. Loeb, L. A. et al. Lethal mutagenesis of HIV with mutagenic nucleoside analogs. Proc. Natl Acad. Sci. USA 96, 1492–1497 (1999).

    Article  CAS  Google Scholar 

  22. Crotty, S. & Andino, R. Implications of high RNA virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin. Microbes Infect. 4, 1301–1307 (2002).

    Article  CAS  Google Scholar 

  23. Bordería, A. V., Rozen-Gagnon, K. & Vignuzzi, M. Fidelity variants and RNA quasispecies. Curr. Top. Microbiol. Immunol. 392, 1–20 (2015).

    Google Scholar 

  24. Stern, A. et al. Costs and benefits of mutational robustness in RNA viruses. Cell Rep. 8, 1026–1036 (2014).

    Article  CAS  Google Scholar 

  25. Wilke, C. O. Evolution of mutational robustness. Mutat. Res. 522, 3–11 (2003).

    Article  CAS  Google Scholar 

  26. Sanjuán, R., Forment, J. & Elena, S. F. In silico predicted robustness of viroid RNA secondary structures. II. Interaction between mutation pairs. Mol. Biol. Evol. 23, 2123–2130 (2006).

    Article  Google Scholar 

  27. Sanjuán, R., Forment, J. & Elena, S. F. In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations. Mol. Biol. Evol. 23, 1427–1436 (2006).

    Article  Google Scholar 

  28. Schultes, E. A. & Bartel, D. P. One sequence, two ribozymes: implications for the emergence of new ribozyme folds. Science 289, 448–452 (2000).

    Article  CAS  Google Scholar 

  29. Sanjuán, R., Cuevas, J. M., Furió, V., Holmes, E. C. & Moya, A. Selection for robustness in mutagenized RNA viruses. PLoS Genet. 3, e93 (2007).

    Article  Google Scholar 

  30. Graci, J. D. et al. Mutational robustness of an RNA virus influences sensitivity to lethal mutagenesis. J. Virol. 86, 2869–2873 (2012).

    Article  CAS  Google Scholar 

  31. Codoñer, F. M., Daròs, J.-A., Solé, R. V. & Elena, S. F. The fittest versus the flattest: experimental confirmation of the quasispecies effect with subviral pathogens. PLoS Pathog. 2, e136 (2006).

    Article  Google Scholar 

  32. Atkinson, N. J., Witteveldt, J., Evans, D. J. & Simmonds, P. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res. 42, 4527–4545 (2014).

    Article  CAS  Google Scholar 

  33. Le Nouën, C. et al. Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization. Proc. Natl Acad. Sci. USA 111, 13169–13174 (2014).

    Article  Google Scholar 

  34. Mueller, S. et al. Live attenuated influenza virus vaccines by computer-aided rational design. Nat. Biotechnol. 28, 723–726 (2010).

    Article  CAS  Google Scholar 

  35. Burns, C. C. et al. Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region. J. Virol. 80, 3259–3272 (2006).

    Article  CAS  Google Scholar 

  36. Aragonès, L., Guix, S., Ribes, E., Bosch, A. & Pintó, R. M. Fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis a virus capsid. PLoS Pathog. 6, e1000797 (2010).

    Article  Google Scholar 

  37. Nougairede, A. et al. Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells. PLoS Pathog. 9, e1003172 (2013).

    Article  CAS  Google Scholar 

  38. Meng, J., Lee, S., Hotard, A. L. & Moore, M. L. Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes. mBio 5, e01704–14 (2014).

    Article  CAS  Google Scholar 

  39. Gaunt, E. et al. Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection. eLife 5, e12735 (2016).

    Article  Google Scholar 

  40. Archetti, M. Genetic robustness at the codon level as a measure of selection. Gene 443, 64–69 (2009).

    Article  CAS  Google Scholar 

  41. McLachlan, A. D. Tests for comparing related amino-acid sequences. Cytochrome c and cytochrome c 551. J. Mol. Biol. 61, 409–424 (1971).

    Article  CAS  Google Scholar 

  42. Burch, C. L. & Chao, L. Evolvability of an RNA virus is determined by its mutational neighbourhood. Nature 406, 625–628 (2000).

    Article  CAS  Google Scholar 

  43. Plotkin, J. B. & Dushoff, J. Codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza A virus. Proc. Natl Acad. Sci. USA 100, 7152–7157 (2003).

    Article  CAS  Google Scholar 

  44. Kovac, A. et al. Misfolded truncated protein τ induces innate immune response via MAPK pathway. J. Immunol. 187, 2732–2739 (2011).

    Article  CAS  Google Scholar 

  45. Dabaghian, M., Latifi, A. M., Tebianian, M., Dabaghian, F. & Ebrahimi, S. M. A truncated C-terminal fragment of Mycobacterium tuberculosis HSP70 enhances cell-mediated immune response and longevity of the total IgG to influenza A virus M2e protein in mice. Antiviral Res. 120, 23–31 (2015).

    Article  CAS  Google Scholar 

  46. Crowder, S. & Kirkegaard, K. Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat. Genet. 37, 701–709 (2005).

    Article  CAS  Google Scholar 

  47. Gonzalez-Lopez, C., Arias, A., Pariente, N., Gómez-Mariano, G. & Domingo, E. Preextinction viral RNA can interfere with infectivity. J. Virol. 78, 3319–3324 (2004).

    Article  CAS  Google Scholar 

  48. Yount, J. S., Kraus, T. A., Horvath, C. M., Moran, T. M. & López, C. B. A novel role for viral-defective interfering particles in enhancing dendritic cell maturation. J. Immunol. 177, 4503–4513 (2006).

    Article  CAS  Google Scholar 

  49. Tapia, K. et al. Defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity. PLoS Pathog. 9, e1003703 (2013).

    Article  Google Scholar 

  50. Boge, T. et al. A probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials. Vaccine 27, 5677–5684 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank M. Declercq, M. Barbet, S. Van der Werf, C. Saleh and A. Pizzorno for assistance and discussions. This work was supported by an Institut Pasteur Innovative Ideas in Vaccinology - GPF Vaccinology grant, an ERC PoC grant no. 727758 and the Roux–Cantarini Fellowship (G.M.).

Author information

Author notes

  1. Rasmus Henningsson and Cyril Barbezange: These authors contributed equally to this work.

Authors and Affiliations

  1. Viral Populations and Pathogenesis Unit, Institut Pasteur, CNRS UMR 3569, 28 rue du Dr. Roux, 75724 Paris cedex 15, France

    Gonzalo Moratorio, Rasmus Henningsson, Cyril Barbezange, Lucia Carrau, Antonio V. Bordería, Hervé Blanc, Stephanie Beaucourt, Enzo Z. Poirier, Thomas Vallet, Bryan C. Mounce & Marco Vignuzzi

  2. International Group for Data Analysis, Institut Pasteur, C3BI, USR 3756 IP CNRS, 28 rue du Dr. Roux, 75724 Paris cedex 15, France

    Rasmus Henningsson, Antonio V. Bordería, Jeremy Boussier & Magnus Fontes

  3. Centre for Mathematical Sciences, Lund University, 22100 Lund, Sweden

    Rasmus Henningsson & Magnus Fontes

  4. Sorbonne Paris Cité, Université Paris Diderot, Cellule Pasteur, 75013 Paris, France

    Lucia Carrau & Enzo Z. Poirier

  5. Unité d'Immunobiologie des Cellules Dendritiques, Institut Pasteur, Inserm 1223, 25 rue du Dr. Roux, 75724 Paris cedex 15, Paris, France

    Jeremy Boussier

  6. Ecole doctorale Frontières du vivant, Université Paris Diderot, 75013 Paris, France

    Jeremy Boussier

Authors
  1. Gonzalo Moratorio
    View author publications

    Search author on:PubMed Google Scholar

  2. Rasmus Henningsson
    View author publications

    Search author on:PubMed Google Scholar

  3. Cyril Barbezange
    View author publications

    Search author on:PubMed Google Scholar

  4. Lucia Carrau
    View author publications

    Search author on:PubMed Google Scholar

  5. Antonio V. Bordería
    View author publications

    Search author on:PubMed Google Scholar

  6. Hervé Blanc
    View author publications

    Search author on:PubMed Google Scholar

  7. Stephanie Beaucourt
    View author publications

    Search author on:PubMed Google Scholar

  8. Enzo Z. Poirier
    View author publications

    Search author on:PubMed Google Scholar

  9. Thomas Vallet
    View author publications

    Search author on:PubMed Google Scholar

  10. Jeremy Boussier
    View author publications

    Search author on:PubMed Google Scholar

  11. Bryan C. Mounce
    View author publications

    Search author on:PubMed Google Scholar

  12. Magnus Fontes
    View author publications

    Search author on:PubMed Google Scholar

  13. Marco Vignuzzi
    View author publications

    Search author on:PubMed Google Scholar

Contributions

G.M., C.B. and M.V. designed the experiments. G.M., C.B., L.C., A.V.B., H.B., E.Z.P., S.B., T.V. and B.C.M. performed experiments. G.M., R.H., J.B., C.B., M.F. and M.V. analysed the data. G.M. and M.V. wrote the paper.

Corresponding author

Correspondence to Marco Vignuzzi.

Ethics declarations

Competing interests

The Institut Pasteur has filed a European Patent Application that covers the alteration of RNA virus sequence space and in particular the manipulation of codons to skew towards non-sense mutation targets such as Stop mutations, as a means of attenuating viruses. G.M., C.B. and M.V. are listed inventors (World Patent Application no. WO2016120412).

Supplementary information

Supplementary Information

Supplementary Figures 1–4, Supplementary Methods, Supplementary References (PDF 8325 kb)

Supplementary Table 1

List of codon changes introduced in Coxsackie virus B3 and influenza A viruses. The table indicates the starting nucleotide position and identity of each codon in the Coxsackie virus B3 P1 region and the influenza A virus PA and HA genes, for wild-type and 1-to-Stop viruses for both serine (S) and leucine (L). (XLSX 16 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moratorio, G., Henningsson, R., Barbezange, C. et al. Attenuation of RNA viruses by redirecting their evolution in sequence space. Nat Microbiol 2, 17088 (2017). https://doi.org/10.1038/nmicrobiol.2017.88

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.88

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology