Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection

Journal name:
Nature
Volume:
505,
Pages:
432–435
Date published:
DOI:
doi:10.1038/nature12816
Received
Accepted
Published online

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 2.4Å resolution crystal structure of the in vitro assembled H protein’s central domain consists of a 170Å-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.

At a glance

Figures

  1. The structure of the H-protein coiled-coil tube.
    Figure 1: The structure of the H-protein coiled-coil tube.

    a, Ribbon representation of the structure. The monomers are individually coloured. b, Top view of the structure with the N terminus closest to the viewer. c, The inner surface of the H-protein tube is coloured according to the electrostatic potential. Blue and red colours correspond to positive and negative potential of 5kT/e, respectively. d, The top view of the tube surface (the orientation of the tube is the same as in b). e, f, Superposition of the H-protein tube (cyan) and the bacteriophage fd capsid (purple), showing that the H protein has a similar inner diameter to fd (Protein Data Bank accession number 2HI5)27.

  2. Helical wheel representations summarizing the inter-helical contacts in different parts of the helical barrel.
    Figure 2: Helical wheel representations summarizing the inter-helical contacts in different parts of the helical barrel.

    a, The 7/2 heptad repeats of domain B. b, The 11/3 hendecad repeats of domain A. The amino-acid sequences arranged in 11- and 7-residue repeats are given on the right. The residues that were mutated to disrupt formation of the H tube are coloured blue. Residues facing the interior are coloured orange. The inward-facing residues are highlighted in green. c, Stereo view of the residues lining the tube centre.

  3. Cryo-electron micrographic tomogram of the [Phi]X174-like phage ST-1 infecting E. coli mini cells.
    Figure 3: Cryo-electron micrographic tomogram of the ΦX174-like phage ST-1 infecting E. coli mini cells.

    ac, Slices of tomograms showing three states of the infection process. dh, Enlarged images taken from ac. d, The virus has attached to the outer membrane (OM). One of the pentameric spikes of an icosahedral particle has recognized a lipopolysaccharide (LPS) molecule in the outer membrane of the E. coli cell wall. e, f, After attachment, the virus extrudes a tube for DNA penetration. A tube can be seen (white arrow) crossing the periplasmic space, lodged in the outer and inner membrane (IM). g, h, After DNA has been injected into the cell, the extended tail starts to disassemble. i, Schematic model of ΦX174 infection.

  4. In vivo and in vitro analysis of the mutant H protein.
    Extended Data Fig. 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 4h. 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.

  5. Simulation of cryo-electron micrographs of [Phi]X174 containing one H tube.
    Extended Data Fig. 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.

  6. Sequence alignment of the [Phi]X174 H and ST-1 H proteins.
    Extended Data Fig. 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.

  7. Coiled-coil structures of [Phi]X174 H protein and P22 portal protein.
    Extended Data Fig. 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.

  8. Sequence alignment of the T7 tail-tube extension protein gp14 with the [Phi]X174 H-protein coiled-coil domain.
    Extended Data Fig. 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.

Accession codes

Referenced accessions

Protein Data Bank

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Author information

  1. These authors contributed equally to this work.

    • Lei Sun,
    • Lindsey N. Young &
    • Xinzheng Zhang

Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA

    • Lei Sun,
    • Xinzheng Zhang,
    • Sergei P. Boudko,
    • Andrei Fokine,
    • Erica Zbornik &
    • Michael G. Rossmann
  2. School of Plant Sciences and the BIO5 Institute, University of Arizona, Tucson, Arizona 85721, USA

    • Lindsey N. Young,
    • Aaron P. Roznowski &
    • Bentley A. Fane
  3. Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA

    • Ian J. Molineux
  4. Present address: The Research Department, Shriner’s Hospital for Children, Portland, Oregon 97239, USA.

    • Sergei P. Boudko

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

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

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: In vivo and in vitro analysis of the mutant H protein. (130 KB)

    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 4h. 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.

  2. Extended Data Figure 2: Simulation of cryo-electron micrographs of ΦX174 containing one H tube. (470 KB)

    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.

  3. Extended Data Figure 3: Sequence alignment of the ΦX174 H and ST-1 H proteins. (565 KB)

    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.

  4. Extended Data Figure 4: Coiled-coil structures of ΦX174 H protein and P22 portal protein. (533 KB)

    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.

  5. Extended Data Figure 5: Sequence alignment of the T7 tail-tube extension protein gp14 with the ΦX174 H-protein coiled-coil domain. (96 KB)

    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.

Supplementary information

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  1. Supplementary Tables (296 KB)

    This file contains Supplementary Tables 1-4.

Additional data