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.

  • Letter
  • Published:

A proofreading-impaired herpesvirus generates populations with quasispecies-like structure

Nature Microbiology volume 4, pages 2175–2183 (2019)Cite this article

Subjects

An Author Correction to this article was published on 01 October 2019

Abstract

RNA virus populations are composed of highly diverse individuals that form a cloud of related sequences commonly referred to as a ‘quasispecies’1,2,3. This diversity arises as a consequence of low-fidelity genome replication4,5. By contrast, DNA virus populations contain more uniform individuals with similar fitness6. Genome diversity is often correlated with increased fitness in RNA viruses, while DNA viruses are thought to require more faithful genome replication. During DNA replication, erroneously incorporated bases are removed by a 3′-5′ exonuclease, a highly conserved enzymatic function of replicative DNA but not RNA polymerases. This proofreading process enhances replication fidelity and ensures the genome integrity of DNA organisms, including large DNA viruses7. Here, we show that a herpesvirus can tolerate impaired exonucleolytic proofreading, resulting in DNA virus populations, which, as in RNA viruses8, are composed of highly diverse genotypes of variable individual fitness. This indicates that herpesvirus mutant diversity may compensate for individual fitness loss. Notably, in vivo infection with diverse virus populations results in a marked increase in virulence compared to genetically homogenous parental virus. While we cannot exclude that the increase in virulence is caused by selection of and/or interactions between individual genotypes, our findings are consistent with quasispecies dynamics. Our results contrast with traditional views of DNA virus replication and evolution, and indicate that a substantial increase in population diversity can lead to higher virulence.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Design and properties of MDV DNA polymerase mutants.
Fig. 2: Genetic diversity of quasispecies-like populations in cell culture.
Fig. 3: Genetic diversity of viruses determines disease outcome.
Fig. 4: Genetic diversity of viruses in vivo.

Similar content being viewed by others

Data availability

The SNPs analysed in this study are supplied as Supplementary Tables. Illumina short reads (FASTQ) have been uploaded to the Short Read Archive and can be accessed under Bioproject no. PRJNA553690.

Code availability

The custom code used to analyse the sequencing data is available at the public repository github (https://github.com/NG-viro/Shannon_Entropy).

References

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

    Article  PubMed  CAS  Google Scholar 

  2. Bordería, A. V., Rozen-Gagnon, K. & Vignuzzi, M. Fidelity variants and RNA quasispecies. Curr. Top. Microbiol. Immunol. 392, 303–322 (2016).

    PubMed  Google Scholar 

  3. Domingo, E. & Schuster, P. What is a quasispecies? Historical origins and current scope. Curr. Top. Microbiol. Immunol. 392, 1–22 (2016).

    CAS  PubMed  Google Scholar 

  4. Drake, J. W. The distribution of rates of spontaneous mutation over viruses, prokaryotes, and eukaryotes. Ann. N. Y. Acad. Sci. 870, 100–107 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Dolan, P. T., Whitfield, Z. J. & Andino, R. Mechanisms and concepts in RNA virus population dynamics and evolution. Annu. Rev. Virol. 5, 69–92 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Fornés, J., Tomás Lázaro, J., Alarcón, T., Elena, S. F. & Sardanyés, J. Viral replication modes in single-peak fitness landscapes: a dynamical systems analysis. J. Theor. Biol. 460, 170–183 (2019).

    Article  PubMed  Google Scholar 

  7. Bębenek, A. & Ziuzia-Graczyk, I. Fidelity of DNA replication: a matter of proofreading. Curr. Genet. 64, 985–996 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Villarreal, L. P. & Witzany, G. Rethinking quasispecies theory: from fittest type to cooperative consortia. World J. Biol. Chem. 4, 79–90 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Osterrieder, N., Kamil, J. P., Schumacher, D., Tischer, B. K. & Trapp, S. Marek’s disease virus: from miasma to model. Nat. Rev. Microbiol. 4, 283–294 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Trimpert, J. et al. A phylogenomic analysis of Marek’s disease virus reveals independent paths to virulence in Eurasia and North America. Evol. Appl. 10, 1091–1101 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Szpara, M. L. et al. Evolution and diversity in human herpes simplex virus genomes. J. Virol. 88, 1209–1227 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Weller, S. K. & Coen, D. M. Herpes simplex viruses: mechanisms of DNA replication. Cold Spring Harb. Perspect. Biol. 4, a013011 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Hwang, C. B.-C. in DNA Replication and Related Cellular Processes (ed. Kušić-Tišma, J.) 143–160 (InTech, 2011).

  14. Song, L., Chaudhuri, M., Knopf, C. W. & Parris, D. S. Contribution of the 3′- to 5′-exonuclease activity of herpes simplex virus type 1 DNA polymerase to the fidelity of DNA synthesis. J. Biol. Chem. 279, 18535–18543 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Lawler, J. L. & Coen, D. M. HSV-1 DNA polymerase 3′-5′ exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression. J. Gen. Virol. 99, 1432–1437 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hall, J. D., Orth, K. L., Sander, K. L., Swihart, B. M. & Senese, R. A. Mutations within conserved motifs in the 3′−5′ exonuclease domain of herpes simplex virus DNA polymerase. J. Gen. Virol. 76, 2999–3008 (1995).

    Article  CAS  PubMed  Google Scholar 

  17. Hwang, C. B., Ruffner, K. L. & Coen, D. M. A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance. J. Virol. 66, 1774–1776 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hwang, Y. T., Liu, B. Y., Hong, C. Y., Shillitoe, E. J. & Hwang, C. B. Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo. J. Virol. 73, 5326–5332 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Crotty, S., Cameron, C. E. & Andino, R. RNA virus error catastrophe: direct molecular test by using ribavirin. Proc. Natl Acad. Sci. USA 98, 6895–6900 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Taddei, F. et al. Role of mutator alleles in adaptive evolution. Nature 387, 700–702 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Mansky, L. M. & Cunningham, K. S. Virus mutators and antimutators: roles in evolution, pathogenesis and emergence. Trends Genet. 16, 512–517 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Xu, M., Fitzgerald, S. D., Zhang, H., Karcher, D. M. & Heidari, M. Very virulent plus strains of MDV induce an acute form of transient paralysis in both susceptible and resistant chicken lines. Viral Immunol. 25, 306–323 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Pandey, U. et al. DNA from dust: comparative genomics of large DNA viruses in field surveillance samples. mSphere 1, e00132-16 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Jaramillo, N. et al. Evidence of Muller’s ratchet in herpes simplex virus type 1. J. Gen. Virol. 94, 366–375 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Xiao, Y. et al. RNA recombination enhances adaptability and is required for virus spread and virulence. Cell Host Microbe 19, 493–503 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vignuzzi, M., Stone, J. K., Arnold, J. J., Cameron, C. E. & Andino, R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439, 344–348 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Fitzsimmons, W. J. et al. A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus. PLoS Biol. 16, e2006459 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Holmes, E. C. The RNA virus quasispecies: fact or fiction? J. Mol. Biol. 400, 271–273 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Bull, J. J., Meyers, L. A. & Lachmann, M. Quasispecies made simple. PLoS Comput. Biol. 1, e61 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lauring, A. S. & Andino, R. Quasispecies theory and the behavior of RNA viruses. PLoS Pathog. 6, e1001005 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Leeks, A., Segredo-Otero, E. A., Sanjuán, R. & West, S. A. Beneficial coinfection can promote within-host viral diversity. Virus Evol. 4, vey028 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Jarosinski, K. W., Arndt, S., Kaufer, B. B. & Osterrieder, N. Fluorescently tagged pUL47 of Marek’s disease virus reveals differential tissue expression of the tegument protein in vivo. J. Virol. 86, 2428–2436 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schat, K. A. & Purchase, H. in A Laboratory Manual for the Isolation and Identification of Avian Pathogens (ed. Swayne, D. E.) 223–234 (American Association of Avian Pathologists, 1998).

  36. Longo, P. A., Kavran, J. M., Kim, M.-S. & Leahy, D. J. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529, 227–240 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schumacher, D., Tischer, B. K., Fuchs, W. & Osterrieder, N. Reconstitution of Marek’s disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. J. Virol. 74, 11088–11098 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Schumacher, D., Tischer, B. K., Trapp, S. & Osterrieder, N. The protein encoded by the US3 orthologue of Marek’s disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown. J. Virol. 79, 3987–3997 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tischer, B. K., Smith, G. A. & Osterrieder, N. En passant mutagenesis: a two step markerless red recombination system. Methods Mol. Biol. 634, 421–430 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Blin, N. & Stafford, D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303–2308 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sambrook, J. & Russell, D. W. Molecular Cloning: a Laboratory Manual 3rd edn (Cold Spring Harbor Lab. Press, 2001).

  42. Hirt, B. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369 (1967).

    Article  CAS  PubMed  Google Scholar 

  43. Baigent, S. J. et al. Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek’s disease. J. Gen. Virol. 87, 769–776 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Tischer, B. K., von Einem, J., Kaufer, B. & Osterrieder, N. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques 40, 191–197 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Wu, S. Y. & Chiang, C. M. Expression and purification of epitope-tagged multisubunit protein complexesfrom mammalian cells. Curr. Protoc. Mol. Biol. Chapter 16, Unit 16.13 (2002).

    PubMed  Google Scholar 

  46. Kühn, F. J. & Knopf, C. W. Herpes simplex virus type 1 DNA polymerase. Mutational analysis of the 3′−5′-exonuclease domain. J. Biol. Chem. 271, 29245–29254 (1996).

    Article  PubMed  Google Scholar 

  47. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at https://arxiv.org/pdf/1207.3907.pdf (2012).

  50. R Core Team. R: a language and environment for statistical computing. R package version 3.1.0 (R Foundation for Statistical Computing, 2014).

  51. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Huson, D. H. & Scornavacca, C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst. Biol. 61, 1061–1067 (2012).

    Article  PubMed  Google Scholar 

  53. Zagordi, O., Däumer, M., Beisel, C. & Beerenwinkel, N. Read length versus depth of coverage for viral quasispecies reconstruction. PLoS ONE 7, e47046 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank the animal caretakers of the Robert-von-Ostertag-Haus for their expert assistance. J.T. was supported by a stipend from the Studienstiftung des Deutschen Volkes.

Author information

Authors and Affiliations

  1. Institut für Virologie, Freie Universität Berlin, Berlin, Germany

    Jakob Trimpert, Nicole Groenke, Dusan Kunec, Kathrin Eschke & Nikolaus Osterrieder

  2. Institut für Biologie, Freie Universität Berlin, Berlin, Germany

    Shulin He & Dino P. McMahon

  3. Department for Materials and Environment, BAM Federal Institute for Materials Research and Testing, Berlin, Germany

    Shulin He & Dino P. McMahon

Authors
  1. Jakob Trimpert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicole Groenke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dusan Kunec
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kathrin Eschke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shulin He
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dino P. McMahon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nikolaus Osterrieder
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.T. performed and analysed the virological, genetic, biochemical, sequencing and animal experiments. N.G. performed the sequence analyses. S.H. and D.P.M. performed the phylogenetic analyses. K.E. and N.O. determined some of the virus growth properties. D.P.M., D.K. and N.O. supervised the study. J.T., D.P.M. and N.O. wrote the paper.

Corresponding author

Correspondence to Nikolaus Osterrieder.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Supplementary Tables 1–12.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Trimpert, J., Groenke, N., Kunec, D. et al. A proofreading-impaired herpesvirus generates populations with quasispecies-like structure. Nat Microbiol 4, 2175–2183 (2019). https://doi.org/10.1038/s41564-019-0547-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0547-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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