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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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.).
The authors declare no competing financial interests.
Extended data figures and tables
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.
a–f, 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.
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.
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). https://doi.org/10.1038/nature12816
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