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The structure of the influenza A virus genome

Nature Microbiology volume 4, pages 1781–1789 (2019)Cite this article

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Abstract

Influenza A viruses (IAVs) constitute a major threat to human health. The IAV genome consists of eight single-stranded viral RNA segments contained in separate viral ribonucleoprotein (vRNP) complexes that are packaged together into a single virus particle. The structure of viral RNA is believed to play a role in assembling the different vRNPs into budding virions1,2,3,4,5,6,7,8 and in directing reassortment between IAVs9. Reassortment between established human IAVs and IAVs harboured in the animal reservoir can lead to the emergence of pandemic influenza strains to which there is little pre-existing immunity in the human population10,11. While previous studies have revealed the overall organization of the proteins within vRNPs, characterization of viral RNA structure using conventional structural methods is hampered by limited resolution and an inability to resolve dynamic components12,13. Here, we employ multiple high-throughput sequencing approaches to generate a global high-resolution structure of the IAV genome. We show that different IAV genome segments acquire distinct RNA conformations and form both intra- and intersegment RNA interactions inside influenza virions. We use our detailed map of IAV genome structure to provide direct evidence for how intersegment RNA interactions drive vRNP cosegregation during reassortment between different IAV strains. The work presented here is a roadmap both for the development of improved vaccine strains and for the creation of a framework to ‘risk assess’ reassortment potential to better predict the emergence of new pandemic influenza strains.

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Fig. 1: Analysis of the IAV genome structure using SHAPE-MaP.
Fig. 2: Intersegment RNA interactions in the IAV genome.
Fig. 3: RNA interactions form a redundant, plastic network to accommodate variation and reassortment.
Fig. 4: Intersegment RNA interactions drive IAV segment cosegregation during reassortment.

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Data availability

All sequencing data have been deposited with the Sequence Read Archive (accession numbers: SRP127020, SRP126994, SRP150669, SRP150677 and SRP212811); the processed SHAPE-MaP reactivities are available in SNRNASM format as Supplementary Table 1 and processed SPLASH data as Supplementary Table 2.

References

  1. Gerber, M., Isel, C., Moules, V. & Marquet, R. Selective packaging of the influenza A genome and consequences for genetic reassortment. Trends Microbiol. 22, 446–455 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Hutchinson, E. C., von Kirchbach, J. C., Gog, J. R. & Digard, P. Genome packaging in influenza A virus. J. Gen. Virol. 91, 313–328 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Gavazzi, C. et al. A functional sequence-specific interaction between influenza A virus genomic RNA segments. Proc. Natl Acad. Sci. USA 110, 16604–16609 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Fournier, E. et al. A supramolecular assembly formed by influenza A virus genomic RNA segments. Nucleic Acids Res. 40, 2197–2209 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Marsh, G. A., Rabadán, R., Levine, A. J. & Palese, P. Highly conserved regions of influenza A virus polymerase gene segments are critical for efficient viral RNA packaging. J. Virol. 82, 2295–2304 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Gog, J. R. et al. Codon conservation in the influenza A virus genome defines RNA packaging signals. Nucleic Acids Res. 35, 1897–1907 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hutchinson, E. C., Curran, M. D., Read, E. K., Gog, J. R. & Digard, P. Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus. J. Virol. 82, 11869–11879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hutchinson, E. C., Wise, H. M., Kudryavtseva, K., Curran, M. D. & Digard, P. Characterisation of influenza A viruses with mutations in segment 5 packaging signals. Vaccine 27, 6270–6275 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gilbertson, B. et al. Influenza NA and PB1 gene segments interact during the formation of viral progeny: localization of the binding region within the PB1 gene. Viruses 8, E238 (2016).

    Article  PubMed  CAS  Google Scholar 

  10. Lowen, A. C. Constraints, drivers, and implications of influenza A virus reassortment. Annu. Rev. Virol. 4, 105–121 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Taubenberger, J. K. & Kash, J. C. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7, 440–451 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Arranz, R. et al. The structure of native influenza virion ribonucleoproteins. Science 338, 1634–1637 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Moeller, A., Kirchdoerfer, R. N., Potter, C. S., Carragher, B. & Wilson, I. A. Organization of the influenza virus replication machinery. Science 338, 1631–1634 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Te Velthuis, A. J. & Fodor, E. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis. Nat. Rev. Microbiol. 14, 479–493 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pflug, A., Lukarska, M., Resa-Infante, P., Reich, S. & Cusack, S. Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Res. 234, 103–117 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Wilkinson, K. A. et al. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. PLoS Biol. 6, e96 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Smola, M. J., Rice, G. M., Busan, S., Siegfried, N. A. & Weeks, K. M. Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat. Protoc. 10, 1643–1669 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Watts, J. M. et al. Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460, 711–716 (2009).

    Article  PubMed Central  CAS  Google Scholar 

  19. Lee, N. et al. Genome-wide analysis of influenza viral RNA and nucleoprotein association. Nucleic Acids Res. 45, 8968–8977 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Williams, G. D. et al. Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication. Nat. Commun. 9, 465 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Baudin, F., Bach, C., Cusack, S. & Ruigrok, R. W. Structure of influenza virus RNP. I. influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. EMBO J. 13, 3158–3165 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kobayashi, Y. et al. Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production. RNA Biol. 13, 883–894 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Gultyaev, A. P. et al. RNA structural constraints in the evolution of the influenza A virus genome NP segment. RNA Biol. 11, 942–952 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Aw, J. G. A. et al. In vivo mapping of eukaryotic RNA interactomes reveals principles of higher-order organization and regulation. Mol. Cell 62, 603–617 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. Cobbin, J. C., Verity, E. E., Gilbertson, B. P., Rockman, S. P. & Brown, L. E. The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus. J. Virol. 87, 5577–5585 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cobbin, J. C. et al. Influenza virus PB1 and neuraminidase gene segments can cosegregate during vaccine reassortment driven by interactions in the PB1 coding region. J. Virol. 88, 8971–8980 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Moreira, É. A. et al. A conserved influenza A virus nucleoprotein code controls specific viral genome packaging. Nat. Commun. 7, 12861 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Saira, K. et al. Sequence analysis of in vivo defective interfering-like RNA of influenza A H1N1 pandemic virus. J. Virol. 87, 8064–8074 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Noda, T. et al. Three-dimensional analysis of ribonucleoprotein complexes in influenza A virus. Nat. Commun. 3, 639 (2012).

    Article  PubMed  CAS  Google Scholar 

  30. Hutchinson, E. C. et al. Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog. 8, e1002993 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hutchinson, E. C. et al. Conserved and host-specific features of influenza virion architecture. Nat. Commun. 5, 4816 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Mitra, S. Detecting RNA tertiary folding by sedimentation velocity analytical ultracentrifugation. Methods Mol. Biol. 1086, 265–288 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Turner, R., Shefer, K. & Ares, M. Jr. Safer one-pot synthesis of the ‘SHAPE’ reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19, 1857–1863 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wilkinson, K. A., Merino, E. J. & Weeks, K. M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat. Protoc. 1, 1610–1616 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Aw, J. G. A., Shen, Y., Nagarajan, N. & Wan, Y. Mapping RNA-RNA interactions globally using biotinylated psoralen. J. Vis. Exp. 123, 55255 (2017).

    Google Scholar 

  36. Jiang, H., Lei, R., Ding, S. & Zhu, S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15, 182 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Busan, S. & Weeks, K. M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143–148 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Gu, Z., Gu, L., Eils, R., Schlesner, M. & Brors, B. circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811–2812 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Busch, A., Richter, A. S. & Backofen, R. IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions. Bioinformatics 24, 2849–2856 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Leontis, N. B. & Zirbel, C. L. in RNA 3D Structure Analysis and Prediction (eds Leontis, N. & Westhof, E.) 281–298 (Springer, 2012).

  42. Natchiar, S. K., Myasnikov, A. G., Kratzat, H., Hazemann, I. & Klaholz, B. P. Visualization of chemical modifications in the human 80S ribosome structure. Nature 551, 472–477 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Nguyen, T. H. D. et al. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Te Velthuis, A. J. W. et al. Mini viral RNAs act as innate immune agonists during influenza virus infection. Nat. Microbiol. 3, 1234–1242 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl Acad. Sci. USA 97, 6108–6113 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hoffmann, E., Stech, J., Guan, Y., Webster, R. & Perez, D. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Tannock, G. A., Paul, J. A. & Barry, R. D. Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens. Infect. Immun. 43, 457–462 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank G.G. Brownlee and J. Kenyon for helpful discussions, J. Kenyon, J.G. Aw, and Y. Wan for sharing protocols, J. Robertson for making the 1M7 reagent, J. Sharps for technical assistance and St Jude Children’s Research Hospital for providing the pHW2000 plasmid. This work was supported by a Wellcome Trust studentship (no. 105399/Z/14/Z to B.D.), a UK Biotechnology and Biological Sciences Research Council studentship (no. BB/M011224/1 to M.L.K.), grants from the National Institutes of Health (nos. HL111527, GM101237 and HG008133 to A.L.), a National Health and Medical Research Council of Australia programme grant (no. ID1071916 to L.E.B.), UK Medical Research Council programme grants (nos. MR/K000241/1 and MR/R009945/1 to E.F.) and a Sir Edward Penley Abraham Cephalosporin Junior Research Fellowship to D.L.V.B.

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Author notes

  1. These authors contributed equally: Bernadeta Dadonaite, Brad Gilbertson.

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Bernadeta Dadonaite, Michael L. Knight, Ervin Fodor & David L. V. Bauer

  2. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

    Brad Gilbertson, Sanja Trifkovic, Steven Rockman & Lorena E. Brown

  3. Seqirus Ltd, Parkville, Victoria, Australia

    Steven Rockman

  4. Department of Biology, University of North Carolina, Chapel Hill, NC, USA

    Alain Laederach

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Contributions

B.D. designed, performed and analysed the SHAPE-MaP and SPLASH experiments and associated virus replication and purification work. M.L.K. performed and analysed the SPLASH experiments on reassortant viruses and associated virus replication and purification experiments. D.L.V.B. designed the SPLASH experiments and analysed the resulting data. B.G. designed and performed the competitive reassortant virus rescue experiments, created the reassortant viruses and analysed the data. S.T. and B.G. performed the viral replication experiments with the reassortant viruses. S.R., A.L., L.E.B., E.F. and D.L.V.B. supervised the work. B.D., B.G. and D.L.V.B. wrote the paper with contributions from the other authors.

Corresponding authors

Correspondence to Lorena E. Brown, Ervin Fodor or David L. V. Bauer.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11.

Reporting Summary

Supplementary Table 1

Complete SHAPE-MaP data in SNRNASM format.

Supplementary Table 2

Complete SPLASH data and reference genome sequences.

Supplementary Table 3

Sequences of primers used.

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Dadonaite, B., Gilbertson, B., Knight, M.L. et al. The structure of the influenza A virus genome. Nat Microbiol 4, 1781–1789 (2019). https://doi.org/10.1038/s41564-019-0513-7

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