Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

Abstract

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus2,3,4,5,6, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Structure of epsilon15 bacteriophage.
Figure 2: The tail structure.
Figure 3: Structure of the portal complex and internal core.
Figure 4: Shell protein structure.

References

  1. 1

    McConnell, M., Reznick, A. & Wright, A. Studies on the initial interactions of bacteriophage epsilon15 with its host cell, Salmonella anatum. Virology 94, 10–23 (1979)

    CAS  Article  Google Scholar 

  2. 2

    Jiang, W. et al. Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions. Nature Struct. Biol. 10, 131–135 (2003)

    CAS  Article  Google Scholar 

  3. 3

    Wikoff, W. R. et al. Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289, 2129–2133 (2000)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Fokine, A. et al. Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry. Proc. Natl Acad. Sci. USA 102, 7163–7168 (2005)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Morais, M. C. et al. Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29. Mol. Cell 18, 149–159 (2005)

    CAS  Article  Google Scholar 

  6. 6

    Baker, M. L., Jiang, W., Rixon, F. J. & Chiu, W. Common ancestry of herpesviruses and tailed DNA bacteriophages. J. Virol. 79, 14967–14970 (2005)

    CAS  Article  Google Scholar 

  7. 7

    Breitbart, M., Rohwer, F. & Abedon, S. T. in Phages: their Role in Bacterial Pathogenesis and Biotechnology (eds Waldor, M. K., Friedman, D. I. & Adhya, S. L.) 66–91 (ASM Press, Washington DC, 2005)

    Google Scholar 

  8. 8

    Bazinet, C. & King, J. The DNA translocating vertex of dsDNA bacteriophage. Annu. Rev. Microbiol. 39, 109–129 (1985)

    CAS  Article  Google Scholar 

  9. 9

    Smith, D. E. et al. The bacteriophage straight phi29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Earnshaw, W. C., King, J., Harrison, S. C. & Eiserling, F. A. The structural organization of DNA packaged within the heads of T4 wild-type, isometric and giant bacteriophages. Cell 14, 559–568 (1978)

    CAS  Article  Google Scholar 

  11. 11

    Arsuaga, J., Tan, R. K., Vazquez, M., Sumners de, W. & Harvey, S. C. Investigation of viral DNA packaging using molecular mechanics models. Biophys. Chem. 101–102, 475–484 (2002)

    Article  Google Scholar 

  12. 12

    Zhang, Z. et al. Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy. J. Mol. Biol. 297, 615–626 (2000)

    CAS  Article  Google Scholar 

  13. 13

    Cerritelli, M. E. et al. Encapsidated conformation of bacteriophage T7 DNA. Cell 91, 271–280 (1997)

    CAS  Article  Google Scholar 

  14. 14

    Lurz, R. et al. Structural organisation of the head-to-tail interface of a bacterial virus. J. Mol. Biol. 310, 1027–1037 (2001)

    CAS  Article  Google Scholar 

  15. 15

    Carazo, J. M., Fujisawa, H., Nakasu, S. & Carrascosa, J. L. Bacteriophage T3 gene 8 product oligomer structure. J. Ultrastruct. Mol. Struct. Res. 94, 105–113 (1986)

    CAS  Article  Google Scholar 

  16. 16

    Simpson, A. A. et al. Structure of the bacteriophage phi29 DNA packaging motor. Nature 408, 745–750 (2000)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Orlova, E. V. et al. Structure of a viral DNA gatekeeper at 10 Å resolution by cryo-electron microscopy. EMBO J. 22, 1255–1262 (2003)

    CAS  Article  Google Scholar 

  18. 18

    Agirrezabala, X. et al. Structure of the connector of bacteriophage T7 at 8 Å resolution: structural homologies of a basic component of a DNA translocating machinery. J. Mol. Biol. 347, 895–902 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Tang, L., Marion, W. R., Cingolani, G., Prevelige, P. E. & Johnson, J. E. Three-dimensional structure of the bacteriophage P22 tail machine. EMBO J. 24, 2087–2095 (2005)

    CAS  Article  Google Scholar 

  20. 20

    Cerritelli, M. E. et al. A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core. J. Mol. Biol. 327, 1–6 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Molineux, I. J. No syringes please, ejection of phage T7 DNA from the virion is enzyme driven. Mol. Microbiol. 40, 1–8 (2001)

    CAS  Article  Google Scholar 

  22. 22

    Tavares, P., Lurz, R., Stiege, A., Ruckert, B. & Trautner, T. A. Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1. J. Mol. Biol. 264, 954–967 (1996)

    CAS  Article  Google Scholar 

  23. 23

    Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988)

    CAS  Article  Google Scholar 

  24. 24

    Booth, C. R. et al. A 9 Å single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope. J. Struct. Biol. 147, 116–127 (2004)

    Article  Google Scholar 

  25. 25

    Jiang, W. et al. Semi-automated icosahedral particle reconstruction at sub-nanometer resolution. J. Struct. Biol. 136, 214–225 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    CAS  Article  Google Scholar 

  27. 27

    Jiang, W., Baker, M. L., Ludtke, S. J. & Chiu, W. Bridging the information gap: computational tools for intermediate resolution structure interpretation. J. Mol. Biol. 308, 1033–1044 (2001)

    CAS  Article  Google Scholar 

  28. 28

    Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We acknowledge the support of grants from National Institutes of Health and the Robert Welch Foundation. We thank M. Dougherty for the production of the animations, M. Baker for the AIRS program for secondary structure element identification, and M. F. Schmid and F. Rixon for discussions. Author Contributions W.J. developed the image processing methods and solved and analysed the structures; J.C. and J.J. collected the 200- and 300-kV image data respectively; P.W. did the biochemical preparation and analysis; and W.J., P.W., J.K. and W.C. interpreted the structure and wrote the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Wah Chiu.

Ethics declarations

Competing interests

The three-dimensional density maps have been deposited into the EBI-MSD EMD database with accession codes EMD-1175 for the complete structure without symmetry imposition and EMD-1176 for the icosahedral shell structure. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

This file contains additional details on the methods used in this study, including purification of Epsilon15 phage, CryoEM imaging, 3-D icosahedral reconstruction 3-D non-icosahedral reconstruction and a structural analysis. (DOC 46 kb)

Supplementary Figures

Supplementary Figures 1–8 with accompanying legends. (PPT 12200 kb)

Supplementary Movie 1

The complete structure of Epsilon15 phage showing each of the structural components including tailspikes, tail hub, portal, core, dsDNA and shell proteins based on the 20 Å reconstruction with no symmetry imposed. (MPG 33850 kb)

Supplementary-Movie 2

The icosahedral reconstruction of Epsilon15 phage at 9.5 Å resolution showing the locations of the α-helices and β-sheets of the average capsid shell protein. (MPG 35257 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jiang, W., Chang, J., Jakana, J. et al. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439, 612–616 (2006). https://doi.org/10.1038/nature04487

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

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