Structures of T7 bacteriophage portal and tail suggest a viral DNA retention and ejection mechanism

Abstract

Double-stranded DNA bacteriophages package their genome at high pressure inside a procapsid through the portal, an oligomeric ring protein located at a unique capsid vertex. Once the DNA has been packaged, the tail components assemble on the portal to render the mature infective virion. The tail tightly seals the ejection conduit until infection, when its interaction with the host membrane triggers the opening of the channel and the viral genome is delivered to the host cell. Using high-resolution cryo-electron microscopy and X-ray crystallography, here we describe various structures of the T7 bacteriophage portal and fiber-less tail complex, which suggest a possible mechanism for DNA retention and ejection: a portal closed conformation temporarily retains the genome before the tail is assembled, whereas an open portal is found in the tail. Moreover, a fold including a seven-bladed β-propeller domain is described for the nozzle tail protein.

Fig. 1

Structure of the T7 bacteriophage portal. a Ribbon representation of the gp8_closed structure of the portal with rainbow coloring by monomer. The dimensions of the particle are indicated. Left, lateral view; right, axial view. b Ribbon representation of a monomer of the gp8_closed structure, colored by domains, with relevant secondary structure elements and structural features indicated. c Electrostatic potential on the external (up) and inner channel (down) surfaces of the gp8_closed portal. A thin positively charged channel ring, formed by R368, is indicated. d Detail of the superposition of gp8_closed (blue ribbon) and gp8_open structures (orange ribbon). The conformational change of the channel valve is indicated with an arrow and maximum torsion angle is shown. e Comparison of the two conformations by showing two opposed monomers. Left, closed conformation shown in blue ribbon. Right, open conformation shown in orange ribbon. The crown domain is not visible for the open conformation and has not been shown for the closed connector for the sake of clarity

Fig. 2

Structure of the T7 tail. a Ribbon representation of the cryo-EM T7 tail structure (gp8-gp11-gp12). Gp8 portal, gp11 adaptor, and gp12 nozzle proteins are shown in purple, green, and orange, respectively. b Longitudinal cut of the electrostatic potential surface

Fig. 3

Atomic structures of T7 tail proteins. a Superposition of the gp8_open as in the tail complex (orchid purple) and gp8_closed (blue) portal monomers. Distances and angles of the moving regions are indicated. b Ribbon representation of gp11 adaptor protein monomer. The different domains are colored and labeled. c Ribbon representation of gp12 nozzle protein monomer. The different domains are colored and labeled

Fig. 4

Tail portal-adaptor interaction. a Ribbon representation of the gp8 portal and gp11 adaptor proteins in the tail complex. Left, binary gp8-gp11 complex; right, close-up of the interaction region. In order to facilitate the interpretation of the image, only one subunit of the gp11 oligomer is shown in the close-up view. The adaptor subunits are shown in dark green and light blue; the four subunits of gp8 interacting with gp11 subunit highlighted in the right panel are shown in plum, magenta, purple, and pink, whereas the rest of the subunits are indicated in grey. b Surface charge distribution of the portal/adaptor interaction. In order to facilitate the interpretation, a single monomer of gp11 protein is shown. Left, view from the outside the complex showing the portal electrostatic surface and a ribbon representation of gp11. Right, view from the inside of the channel, showing the adaptor electrostatic surface, and a ribbon representation of the portal protomers

Fig. 5

Structural characterization of the nozzle protein. a Electrostatic potential surfaces of the interacting region between the gp11 adaptor (top) and gp12 nozzle (bottom). b Ribbon representation of gp12 protein structure in the tail complex in orange, with the four closing gates highlighted in dark blue. Diameters of the channel at each of the gates when measured from Cα to Cα are as follows: gate 1, 18.3 Å; gate 2, 8.6 Å (from carbonyl O to carbonyl O); gate 3, 13.8 Å; and gate 4, 23 Å. c Detail of the gp12 closing gate 2, with the map density shown in mesh and the protein atomic model in orange ribbon. Left, lateral view; Right, axial view

Fig. 6

Proposed model for T7 bacteriophage DNA securing inside the capsid. a Overlapping of the “free” portal into the prohead (left) and the tail complex into the mature virus (right) from central sections through the reconstructions described in ref. . b Scheme showing the T7 bacteriophage assembly pathway. The capsid is shown in pink, the portal (gp8) in purple, the core complex in light blue, the terminase in gray, the adaptor (gp11) in green, the nozzle (gp12) in orange, the fibers in dark blue, and the DNA in black. The portal channel valve can be either open or closed. When the portal is in the prohead, the terminase–portal interaction stabilizes the open conformation, allowing DNA packaging. When the terminase leaves the complex, the portal channel valve closes, thus preventing DNA leakage from the capsid. The interaction of the portal with the adaptor protein re-establishes the open conformation of the portal channel valve, permitting the DNA to slip along the tail channel up to the nozzle, ready for ejection. In the mature virus, the gates of the nozzle protein close, retaining the DNA in the tail channel. These gates are closed until the reorganization of the nozzle (untwisting), which is triggered by the interaction of the tip and the fibers with the host membrane. The gates then open and the viral genome is ejected