Maturation of phage T7 involves structural modification of both shell and inner core components

Abstract

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

Figure 1

3D reconstruction of the prohead: (A, B) Volume representation of the 3D reconstructed prohead viewed along the longitudinal and orthogonal axes, respectively. The density is displayed to emphasize the characteristic surface corrugation of the capsomers. The left half in each image corresponds to the nonsymmetrized reconstruction (p1), while the right half represents the reconstruction reinforced by five-fold symmetrization along the longitudinal axis of the virus. (C) Projection density maps of central sections of the corresponding reconstructions (p1 and p5), showing the internal core structure extending from the singular vertex of the prohead. Arrows point to presumptive connections between the shell and the scaffold. The bracket outlines the region corresponding to the scaffold layer.

Figure 2

3D reconstruction of the core in proheads. (A) Cross section of the nonsymmetrized prohead viewed along the two-fold axis of symmetry (sectioned surface is shown in gray). The density is displayed at 1σ above the mean to show the scaffolding lattice arrangement. For clarity, the residual pentameric density has been computationally removed. (B) Rotational analysis of the harmonic components of the plane groups shown by color-coded rectangles in (A). A representative plane of each group is shown at the right. The rotational analysis shown in the graphics corresponds to the inner region encircled by the corresponding color codes. The outer radii of the regions selected for the calculation of the rotational harmonics are 100 Å (green), 70 Å (blue), and 65 Å (red), respectively. The vertical axis in the graphics represents the percentage of rotational energy in each harmonic, while the horizontal axis represents the range of analyzed harmonics. (C) Surface representation of the four-fold symmetrized core complex reconstructed after penton-less shell subtraction, and final removal of the scaffolding moiety (see Materials and methods). A side view is shown on the left, and a view down the five-fold axis toward the particle base on the right. (D) Docking of the previously reported 8 Å resolution connector structure (Agirrezabala et al, 2005) into the reconstructed core complex. A cross section is shown on the left, and the bottom view on the right.

Figure 3

Organization of the prohead. (A) Sectioned half volume of fitted 3D reconstructions of the five-fold symmetrized shell (displayed at σ=3.2 to enhance the corrugations on the inner face of the shell, yellow), the four-fold symmetrized core (magenta), and the previously described connector (Agirrezabala et al, 2005) (green). The nonsymmetrized core–scaffolding complex (transparent gray representation) is displayed at high contour level (1σ) to facilitate visualization of molecular boundaries. (B) Close-up perspective view of the nonsectioned superimposed reconstructions shown in (A). This view is slightly tilted from the view in (A) to highlight the connections between the scaffold lattice and the core, at the level of the wings of the connector. Arrows point to connection points. Inset: view of the nubbins of density in the inner face of the shell. (C) Surface representation of the core–scaffold reconstruction after shell subtraction. No symmetry was imposed. The colored stripes and the long arrows highlight the suggested network arrangement of the scaffolding subunits.

Figure 4

3D reconstruction of the mature virus. (A) Surface-shaded representation of the five-fold symmetrized complete virion viewed along a two-fold axis of symmetry. (B) Central section of the corresponding reconstruction. In this perpendicular view to the connector–core axis, the DNA projects punctuate patterns spaced 2–2.5 nm. The central, less ordered density probably corresponds to the last packaged segment of DNA, suggesting that it is collapsed on itself. (C) Central section viewed along the five-fold axis of symmetry to show the concentric pattern of the packaged DNA. The right-hand half shows the rotationally averaged section. Inset: The radial density plot of this section exhibits an outer dense peak corresponding to the viral capsid, and then at least six equally spaced rings. Three additional wider peaks could also be noted towards the inner radius. (D) Enlarged perspective view of the proximal part of the tail shown in (A), and (E) central section after the local six-fold symmetrization of this domain. This allows to highlight the density corresponding to the partially reconstructed fiber attachment proteins (the densities protruding outside from the equatorial region of the tail, see the stars). The DNA is placed along the core–tail axis. Arrows mark the position where the channel is fully closed.

Figure 5

Domain model for the core rearrangement during viral maturation. (A) Gray level central sections of the core region reconstructed from proheads (left) and final virus (right). (B) Schematic model of the core structural transition showing the central sections of the reconstructed prohead (left) and the mature virus (right). The different domains are coded using different colors: connector, green; wider toroid, blue; eight-folded toroid, yellow; and upper cylinder, red. The arrows represent the possible direction of the movements of the complex. The assignment of the structural proteins to each of the domains is based in the predominant rotational symmetry of the domains determined in this work and the previously proposed stochiometry (Cerritelli et al, 2003b). (C) 3D structure of the prohead (left) and the mature virus (right) shown along the longitudinal core–vertex five-fold symmetry axis. The connector structure (highlighted in green) is visible through the open pentameric vertex of the prohead and mature virus capsid reconstructions.