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Highly heterogeneous mutation rates in the hepatitis C virus genome

Nature Microbiology volume 1, Article number: 16045 (2016) Cite this article

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Abstract

Spontaneous mutations are the ultimate source of genetic variation and have a prominent role in evolution. RNA viruses such as hepatitis C virus (HCV) have extremely high mutation rates, but these rates have been inferred from a minute fraction of genome sites, limiting our view of how RNA viruses create diversity. Here, by applying high-fidelity ultradeep sequencing to a modified replicon system, we scored >15,000 spontaneous mutations, encompassing more than 90% of the HCV genome. This revealed >1,000-fold differences in mutability across genome sites, with extreme variations even between adjacent nucleotides. We identify base composition, the presence of high- and low-mutation clusters and transition/transversion biases as the main factors driving this heterogeneity. Furthermore, we find that mutability correlates with the ability of HCV to diversify in patients. These data provide a site-wise baseline for interrogating natural selection, genetic load and evolvability in HCV, as well as for evaluating drug resistance and immune evasion risks.

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Figure 1: Experimental system for the accumulation of spontaneous mutations in HCV sequences.
Figure 2: HCV genome-wide mutation frequency.
Figure 3: Relationship between replicon mutation frequencies and genetic diversity in patients.
Figure 4: HCV genome-wide mutational bias.

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References

  1. Elde, N. C. et al. Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses. Cell 150, 831–841 (2012).

    Article  Google Scholar 

  2. Goldberg, D. E., Siliciano, R. F. & Jacobs, W. R. Jr. Outwitting evolution: fighting drug-resistant TB, malaria, and HIV. Cell 148, 1271–1283 (2012).

    Article  Google Scholar 

  3. Rosenberg, R. Detecting the emergence of novel, zoonotic viruses pathogenic to humans. Cell Mol. Life Sci. 72, 1115–1125 (2015).

    Article  Google Scholar 

  4. Schotsaert, M. & Garcia-Sastre, A. Influenza vaccines: a moving interdisciplinary field. Viruses 6, 3809–3826 (2014).

    Article  Google Scholar 

  5. Lauring, A. S., Frydman, J. & Andino, R. The role of mutational robustness in RNA virus evolution. Nature Rev. Microbiol. 11, 327–336 (2013).

    Article  Google Scholar 

  6. Andino, R. & Domingo, E. Viral quasispecies. Virology 479–480, 46–51 (2015).

    Article  Google Scholar 

  7. Menéndez-Arias, L. Mutation rates and intrinsic fidelity of retroviral reverse transcriptases. Viruses 1, 1137–1165 (2009).

    Article  Google Scholar 

  8. Harris, R. S. & Dudley, J. P. APOBECs and virus restriction. Virology 479–480, 131–145 (2015).

    Article  Google Scholar 

  9. Smith, E. C. & Denison, M. R. Coronaviruses as DNA wannabes: a new model for the regulation of RNA virus replication fidelity. PLoS Pathogens 9, e1003760 (2013).

    Article  Google Scholar 

  10. Tomaselli, S., Galeano, F., Locatelli, F. & Gallo, A. ADARs and the balance game between virus infection and innate immune cell response. Curr. Issues Mol. Biol. 17, 37–52 (2015).

    Google Scholar 

  11. Sanjuán, R., Nebot, M. R., Chirico, N., Mansky, L. M. & Belshaw, R. Viral mutation rates. J. Virol. 84, 9733–9748 (2010).

    Article  Google Scholar 

  12. Duffy, S., Shackelton, L. A. & Holmes, E. C. Rates of evolutionary change in viruses: patterns and determinants. Nature Rev. Genet. 9, 267–276 (2008).

    Article  Google Scholar 

  13. Biek, R., Pybus, O. G., Lloyd-Smith, J. O. & Didelot, X. Measurably evolving pathogens in the genomic era. Trends Ecol. Evol. 30, 306–313 (2015).

    Article  Google Scholar 

  14. Zhang, X., Rennick, L. J., Duprex, W. P. & Rima, B. K. Determination of spontaneous mutation frequencies in measles virus under nonselective conditions. J. Virol. 87, 2686–2692 (2013).

    Article  Google Scholar 

  15. Gago, S., Elena, S. F., Flores, R. & Sanjuán, R. Extremely high mutation rate of a hammerhead viroid. Science 323, 1308 (2009).

    Article  Google Scholar 

  16. Combe, M. & Sanjuán, R. Variation in RNA virus mutation rates across host cells. PLoS Pathogens 10, e1003855 (2014).

    Article  Google Scholar 

  17. Cuevas, J. M., González-Candelas, F., Moya, A. & Sanjuán, R. The effect of ribavirin on the mutation rate and spectrum of hepatitis C virus in vivo. J. Virol. 83, 5760–5764 (2009).

    Article  Google Scholar 

  18. Cuevas, J. M., Geller, R., Garijo, R., López-Aldeguer, J. & Sanjuán, R. Extremely high mutation rate of HIV-1 in vivo. PLoS Biol. 13, e1002251 (2015).

    Article  Google Scholar 

  19. Ribeiro, R. M. et al. Quantifying the diversification of hepatitis C virus (HCV) during primary infection: estimates of the in vivo mutation rate. PLoS Pathogens 8, e1002881 (2012).

    Article  Google Scholar 

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

    Article  Google Scholar 

  21. Gower, E., Estes, C., Blach, S., Razavi-Shearer, K. & Razavi, H. Global epidemiology and genotype distribution of the hepatitis C virus infection. J. Hepatol. 61, S45–S57 (2014).

    Article  Google Scholar 

  22. Westbrook, R. H. & Dusheiko, G. Natural history of hepatitis C. J. Hepatol. 61, S58–S68 (2014).

    Article  Google Scholar 

  23. Farci, P. et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 288, 339–344 (2000).

    Article  Google Scholar 

  24. Pawlotsky, J. M. Treatment failure and resistance with direct-acting antiviral drugs against hepatitis C virus. Hepatology 53, 1742–1751 (2011).

    Article  Google Scholar 

  25. Bartenschlager, R., Kaul, A. & Sparacio, S. Replication of the hepatitis C virus in cell culture. Antiviral Res. 60, 91–102 (2003).

    Article  Google Scholar 

  26. Lohmann, V. et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999).

    Article  Google Scholar 

  27. Kennedy, S. R. et al. Detecting ultralow-frequency mutations by duplex sequencing. Nature Protoc. 9, 2586–2606 (2014).

    Article  Google Scholar 

  28. Schmitt, M. W. et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 14508–14513 (2012).

    Article  Google Scholar 

  29. Mauger, D. M. et al. Functionally conserved architecture of hepatitis C virus RNA genomes. Proc. Natl Acad. Sci. USA 112, 3692–3697 (2015).

    Google Scholar 

  30. Bankwitz, D. et al. Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes. J. Virol. 84, 5751–5763 (2010).

    Article  Google Scholar 

  31. Taylor, D. R., Puig, M., Darnell, M. E., Mihalik, K. & Feinstone, S. M. New antiviral pathway that mediates hepatitis C virus replicon interferon sensitivity through ADAR1. J. Virol. 79, 6291–6298 (2005).

    Article  Google Scholar 

  32. Powdrill, M. H. et al. Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance. Proc. Natl Acad. Sci. USA 108, 20509–20513 (2011).

    Article  Google Scholar 

  33. Bikard, D. & Marraffini, L. A. Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages. Curr. Opin. Immunol. 24, 15–20 (2012).

    Article  Google Scholar 

  34. Guo, H., Arambula, D., Ghosh, P. & Miller, J. F. Diversity-generating retroelements in phage and bacterial genomes. Microbiol. Spectr. 2, MDNA3-0029-2014 (2014).

    Google Scholar 

  35. Geller, R. et al. The external domains of the HIV-1 envelope are a mutational cold spot. Nature Commun. 6, 8571 (2015).

    Article  Google Scholar 

  36. Duchène, S., Ho, S. Y. & Holmes, E. C. Declining transition/transversion ratios through time reveal limitations to the accuracy of nucleotide substitution models. BMC Evol. Biol. 15, 36 (2015).

    Article  Google Scholar 

  37. Hershberg, R. & Petrov, D. A. Evidence that mutation is universally biased towards AT in bacteria. PLoS Genet. 6, e1001115 (2010).

    Article  Google Scholar 

  38. Zhang, Z. & Gerstein, M. Patterns of nucleotide substitution, insertion and deletion in the human genome inferred from pseudogenes. Nucleic Acids Res. 31, 5338–5348 (2003).

    Article  Google Scholar 

  39. Keller, I., Bensasson, D. & Nichols, R. A. Transition–transversion bias is not universal: a counter example from grasshopper pseudogenes. PLoS Genet. 3, e22 (2007).

    Article  Google Scholar 

  40. Zhu, Y. O., Siegal, M. L., Hall, D. W. & Petrov, D. A. Precise estimates of mutation rate and spectrum in yeast. Proc. Natl Acad. Sci. USA 111, E2310–E2318 (2014).

    Article  Google Scholar 

  41. Ossowski, S. et al. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327, 92–94 (2010).

    Article  Google Scholar 

  42. Pause, A. et al. An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA. J. Biol. Chem. 278, 20374–20380 (2003).

    Article  Google Scholar 

  43. Pietschmann, T., Lohmann, V., Rutter, G., Kurpanek, K. & Bartenschlager, R. Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J. Virol. 75, 1252–1264 (2001).

    Article  Google Scholar 

  44. Binder, M. et al. Replication vesicles are load- and choke-points in the hepatitis C virus lifecycle. PLoS Pathogens 9, e1003561 (2013).

    Article  Google Scholar 

  45. Gosert, R. et al. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J. Virol. 77, 5487–5492 (2003).

    Article  Google Scholar 

  46. Quinkert, D., Bartenschlager, R. & Lohmann, V. Quantitative analysis of the hepatitis C virus replication complex. J. Virol. 79, 13594–13605 (2005).

    Article  Google Scholar 

  47. Moradpour, D., Penin, F. & Rice, C. M. Replication of hepatitis C virus. Nature Rev. Microbiol. 5, 453–463 (2007).

    Article  Google Scholar 

  48. Dahari, H., Ribeiro, R. M., Rice, C. M. & Perelson, A. S. Mathematical modeling of subgenomic hepatitis C virus replication in Huh-7 cells. J. Virol. 81, 750–760 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank R. Bartenschlager for the HCV replicon, F. González-Candelas for the viral RNA, V. Sentandreu and A. Martínez for help with optimization of the Duplex Sequencing technique, R. Flores for suggestions, M. Binder for discussions on HCV replication models and S. Torres for laboratory assistance. This work was financially supported by grants from the European Research Council (ERC-2011-StG- 281191-VIRMUT) and the Spanish MINECO (BFU2013-41329) to R.San.

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Authors and Affiliations

  1. Instituto Cavanilles de Biodiversidad y Biología Evolutiva and Institute for Integrative Systems Biology (I2SysBio), Universitat de València, 46980 Paterna, València, Spain

    Ron Geller, Joan B. Peris, Iván Andreu, Juan-Vicente Bou, Raquel Garijo, José M. Cuevas & Rafael Sanjuán

  2. Unitat de Genómica, Servei Central de Suport a la Investigació Experimental, Universitat de València, 46100 Burjassot, València, Spain

    Úrsula Estada

  3. Regional Center for Biomedical Research, Universidad de Castilla-La Mancha, 02006 Albacete, Spain

    Rosario Sabariegos & Antonio Mas

  4. Departament de Genètica, Universitat de València, 46100 Burjassot, València, Spain

    Rafael Sanjuán

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  1. Ron Geller
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Contributions

R.Ge. performed the experiments, including replicon construction, passaging, Sanger and Duplex Sequencing, and performed the bioinformatics analysis. U.E. and J.M.C. contributed to optimizing the Duplex Sequencing. I.A., J.-V.B. and J.M.C. performed Sanger sequencing of the replicon. R.Ga. performed Sanger sequencing of in vitro polymerization products. R.Sab., J.B.P. and A.M. performed in vitro polymerization assays. R.San. designed and supervised research, acquired funding, analysed the data and wrote the manuscript.

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Correspondence to Rafael Sanjuán.

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The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Tables 1-2, Figures 1-4 and References (PDF 5452 kb)

Supplementary Dataset 1

Sequence of the seven cloned fragments and the fragment used for in vitro polymerization. (TXT 10 kb)

Supplementary Dataset 2

List of mutations obtained by Duplex Sequencing in the three replicon lines, mapped to annotated H77 reference sequence including sequencing coverage, patient sequence diversity, and SHAPE data. (XLSX 2594 kb)

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Geller, R., Estada, Ú., Peris, J. et al. Highly heterogeneous mutation rates in the hepatitis C virus genome. Nat Microbiol 1, 16045 (2016). https://doi.org/10.1038/nmicrobiol.2016.45

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