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Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection


Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls1. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles2,3,4. However, the tail-less, icosahedral, single-stranded DNA ΦX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein5. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host’s periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein’s central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of ten α-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell’s cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.

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Figure 1: The structure of the H-protein coiled-coil tube.
Figure 2: Helical wheel representations summarizing the inter-helical contacts in different parts of the helical barrel.
Figure 3: Cryo-electron micrographic tomogram of the ΦX174-like phage ST-1 infecting E. coli mini cells.

Accession codes


Protein Data Bank

Data deposits

The atomic coordinates and structure factors of ΦX174 H protein have been deposited in the Protein Data Bank under accession numbers 4JPN and 4JPP.


  1. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010)

    PubMed  PubMed Central  Google Scholar 

  2. Molineux, I. J. & Panja, D. Popping the cork: mechanisms of phage genome ejection. Nature Rev. Microbiol. 11, 194–204 (2013)

    CAS  Google Scholar 

  3. Russel, M. & Model, P. in The Bacteriophages (ed. Calendar, R. ) 146–160 (Oxford Univ. Press, 2006)

    Google Scholar 

  4. Peralta, B. et al. Mechanism of membranous tunnelling nanotube formation in viral genome delivery. PLoS Biol. 11, e1001667 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Jazwinski, S. M., Lindberg, A. A. & Kornberg, A. The gene H spike protein of bacteriophages ΦX174 and S13. I. Functions in phage-receptor recognition and in transfection. Virology 66, 283–293 (1975)

    CAS  PubMed  Google Scholar 

  6. Sinsheimer, R. L. A single-stranded DNA from bacteriophage ΦX174. Brookhaven Symp. Biol. 12. 27–34 (1959)

  7. McKenna, R. et al. Atomic structure of single-stranded DNA bacteriophage ΦX174 and its functional implications. Nature 355, 137–143 (1992)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Burgess, A. B. Studies on the proteins of ΦX174. II. The protein composition of the ΦX coat. Proc. Natl Acad. Sci. USA 64, 613–617 (1969)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Leiman, P. G., Chipman, P. R., Kostyuchenko, V. A., Mesyanzhinov, V. V. & Rossmann, M. G. Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 118, 419–429 (2004)

    CAS  PubMed  Google Scholar 

  10. Chang, J. T. et al. Visualizing the structural changes of bacteriophage epsilon15 and its Salmonella host during infection. J. Mol. Biol. 402, 731–740 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hu, B., Margolin, W., Molineux, I. J. & Liu, J. The bacteriophage T7 virion undergoes extensive structural remodeling during infection. Science 339, 576–579 (2013)

    ADS  CAS  PubMed  Google Scholar 

  12. Incardona, N. L. & Selvidge, L. Mechanism of adsorption and eclipse of bacteriophage ΦX174. II. Attachment and eclipse with isolated Escherichia coli cell wall lipopolysaccharide. J. Virol. 11, 775–782 (1973)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Jazwinski, S. M., Marco, R. & Kornberg, A. The gene H spike protein of bacteriophages ΦX174 and S13. II. Relation to synthesis of the parenteral replicative form. Virology 66, 294–305 (1975)

    CAS  PubMed  Google Scholar 

  14. Tusnady, G. E. & Simon, I. The HMMTOP transmembrane topology prediction server. Bioinformatics 17, 849–850 (2001)

    CAS  PubMed  Google Scholar 

  15. Russ, W. P. & Engelman, D. M. The GxxxG motif: a framework for transmembrane helix-helix association. J. Mol. Biol. 296, 911–919 (2000)

    CAS  PubMed  Google Scholar 

  16. Lupas, A., Van Dyke, M. & Stock, J. Predicting coiled-coils from protein sequences. Science 252, 1162–1164 (1991)

    ADS  CAS  PubMed  Google Scholar 

  17. Woolfson, D. N., Bartlett, G. J., Bruning, M. & Thomson, A. R. New currency for old rope: from coiled-coil assemblies to α-helical barrels. Curr. Opin. Struct. Biol. 22, 432–441 (2012)

    CAS  PubMed  Google Scholar 

  18. Crick, F. H. C. The packing of α-helices: simple coiled-coils. Acta Crystallogr. 6, 689–697 (1953)

    CAS  MATH  Google Scholar 

  19. Gruber, M. & Lupas, A. N. Historical review: another 50th anniversary – new periodicities in coiled coils. Trends Biochem. Sci. 28, 679–685 (2003)

    CAS  PubMed  Google Scholar 

  20. Pauling, L., Corey, R. B. & Branson, H. R. The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl Acad. Sci. USA 37, 205–211 (1951)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cherwa, J. E., Jr, Organtini, L. J., Ashley, R. E., Hafenstein, S. L. & Fane, B. A. In vitro assembly of the ΦX174 procapsid from external scaffolding protein oligomers and early pentameric assembly intermediates. J. Mol. Biol. 412, 387–396 (2011)

    CAS  PubMed  Google Scholar 

  22. Bayer, M. E. & Starkey, T. W. The adsorption of bacteriophage ΦX174 and its interaction with Escherichia coli; a kinetic and morphological study. Virology 49, 236–256 (1972)

    CAS  PubMed  Google Scholar 

  23. Sun, S. et al. The structure of the phage T4 DNA packaging motor suggests a mechanism dependent on electrostatic forces. Cell 135, 1251–1262 (2008)

    CAS  PubMed  Google Scholar 

  24. Aoyama, A., Hamatake, R. K. & Hayashi, M. In vitro synthesis of bacteriophage ΦX174 by purified components. Proc. Natl Acad. Sci. USA 80, 4195–4199 (1983)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Benevides, J. M., Stow, P. L., Ilag, L. L., Incardona, N. L. & Thomas, G. J., Jr Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage ΦX174 determined by Raman spectroscopy: a model for ΦX174 DNA packaging. Biochemistry 30, 4855–4863 (1991)

    CAS  PubMed  Google Scholar 

  26. Shepard, W., Cruse, W. B., Fourme, R., de la Fortelle, E. & Prange, T. A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 Å resolution. Structure 6, 849–861 (1998)

    CAS  PubMed  Google Scholar 

  27. Wang, Y. A. et al. The structure of a filamentous bacteriophage. J. Mol. Biol. 361, 209–215 (2006)

    CAS  PubMed  Google Scholar 

  28. Bowes, J. M. & Dowell, C. E. Purification and some properties of bacteriophage ST-1. J. Virol. 13, 53–61 (1974)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Olia, A. S., Prevelige, P. E., Jr, Johnson, J. E. & Cingolani, G. Three-dimensional structure of a viral genome-delivery portal vertex. Nature Struct. Mol. Biol. 18, 597–603 (2011)

    CAS  Google Scholar 

  30. Perez, G. L., Huynh, B., Slater, M. & Maloy, S. Transport of phage P22 DNA across the cytoplasmic membrane. J. Bacteriol. 191, 135–140 (2009)

    CAS  PubMed  Google Scholar 

  31. Lupas, A. Prediction and analysis of coiled-coil structures. Methods Enzymol. 266, 513–525 (1996)

    CAS  PubMed  Google Scholar 

  32. Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997)

    CAS  PubMed  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  34. Tong, L. & Rossmann, M. G. Rotation function calculations with GLRF program. Methods Enzymol. 276, 594–611 (1997)

    CAS  PubMed  Google Scholar 

  35. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  CAS  PubMed  Google Scholar 

  37. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Google Scholar 

  38. McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

    CAS  PubMed  Google Scholar 

  39. Fane, B. A. & Hayashi, M. Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage ΦX174. Genetics 128, 663–671 (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Roof, W. D., Horne, S. M., Young, K. D. & Young, R. slyD, a host gene required for ΦX174 lysis, is related to the FK506-binding protein family of peptidyl-prolyl cis-trans-isomerases. J. Biol. Chem. 269, 2902–2910 (1994)

    CAS  PubMed  Google Scholar 

  41. Fane, B. A., Shien, S. & Hayashi, M. Second-site suppressors of a cold-sensitive external scaffolding protein of bacteriophage ΦX174. Genetics 134, 1003–1011 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Gordon, E. B., Knuff, C. J. & Fane, B. A. Conformational switch-defective ΦX174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation. J. Virol. 86, 9911–9918 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Uchiyama, A. & Fane, B. A. Identification of an interacting coat-external scaffolding protein domain required for both the initiation of ΦX174 procapsid morphogenesis and the completion of DNA packaging. J. Virol. 79, 6751–6756 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Liu, J. et al. Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells. Proc. Natl Acad. Sci. USA 109, E1481–E1488 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996)

    CAS  PubMed  Google Scholar 

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We thank S. Kelly for help in preparing the manuscript. Use of the Advanced Photon Source (Sector 23) was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number DEAC02-06CH11357. This research was supported by National Science Foundation grants MCB-0948399 (to B.A.F.) and MCB-0443899 (to M.G.R.) and US Department of Agriculture Hatch funds to the University of Arizona (to B.A.F.).

Author information

Authors and Affiliations



B.A.F. and M.G.R. developed the concept. L.S., L.N.Y. and X.Z. designed the experiments. L.S. and S.P.D. worked on the cloning, protein purification and crystallization of the H protein. L.S. and A.F. worked on the structure determination and analysis. L.S., L.N.Y. and B.A.F. characterized the mutant data. L.S., X.Z. and B.A.F. produced the tomographic results. B.A.F., I.J.M., E.Z. and A.P.R. contributed effort to protein, virus and cell purification. L.S., M.G.R. and B.A.F. wrote the paper.

Corresponding authors

Correspondence to Michael G. Rossmann or Bentley A. Fane.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 In vivo and in vitro analysis of the mutant H protein.

a, In vivo analysis of particles synthesized in wild-type and mutant-infected cells. Sedimentation profiles of extracts were analysed by rate zonal sedimentation. Lower fractions contained the faster sedimenting material. Curve colours: wild-type (WT), black; mutant 1 (mut1), blue; mutant 2 (mut2), red. Inset: SDS–polyacrylamide gel electrophoresis of peak fractions. The positions of the coat F, DNA pilot H and spike G proteins are indicated with their respective letter designations. b, c, In vitro analysis of wild-type and mutant H-protein fragments (amino acids 143–282). b, Size exclusion results using HiLoad Superdex 200 (16/60). The mutant proteins migrated as lower-order oligomers. c, SDS–polyacrylamide gel electrophoresis of trypsin-digested proteins after 4 h. The wild-type protein produced a smaller stable fragment (indicated by an arrow), whereas the mutant proteins were fully digested. N-terminal sequencing and mass spectroscopy confirmed that the stable wild-type fragment contained residues 143–221.

Extended Data Figure 2 Simulation of cryo-electron micrographs of ΦX174 containing one H tube.

af, Each column (1–8) shows a different orientation of the virus. Each row shows progressively more noise. The top row has no noise and clearly shows the fivefold vertices of the virus and the buried H tube (sideways in column 1 and top view in column 5). g, Micrographs of the actual virus. All evidence of the fivefold spikes has been lost in row f. Similarly, there is no evidence of the spikes in the actual micrographs shown in row g. Thus the micrographs give no hint of whether there is an H tube or partly assembled H tube in the virus.

Extended Data Figure 3 Sequence alignment of the ΦX174 H and ST-1 H proteins.

These two proteins have 55% identical residues and 70% similar residues. Identical residues are highlighted in red. Similar residues are coloured red and boxed with blue.

Extended Data Figure 4 Coiled-coil structures of ΦX174 H protein and P22 portal protein.

a, PDB Blast result using all of the H-protein amino-acid sequence showed that the coiled-coil regions of the H protein had sequence similarity to the P22 portal protein (30% identical residues, 40% similar residues). The conserved residues, which line the tube centre, are highlighted in green. b, Structure of the H-protein coiled-coil region. c, Overall structure of the P22 portal protein (Protein Data Bank accession number 3LJ5)31. Although the H tubes described here are decamers, it is possible, in view of there probably being 12 H proteins in assembled ΦX174 capsids, that the in vivo assembled H tubes could be dodecamers, like the coiled-coil domain of P22 portal protein.

Extended Data Figure 5 Sequence alignment of the T7 tail-tube extension protein gp14 with the ΦX174 H-protein coiled-coil domain.

The conserved glutamines, which line the tube centre, are highlighted in green. The two-amino-acid deletion in the H-protein sequence occurs where the protein transitions between the 11/3 and 7/2 coiled-coil domains.

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Sun, L., Young, L., Zhang, X. et al. Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection. Nature 505, 432–435 (2014).

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