Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Abstract

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

Fig. 1.

The icosahedral structure of the P22 procapsid at 3.8-Å resolution and the interactions between the scaffolding protein and the coat protein. (A) A typical cryo-EM image of the P22 procapsid. (B) A Cα backbone model from residues 10–425 with eight annotated domains for one gp5 subunit derived from the density. The model is colored from blue (N terminus) to red (C terminus). (C) The Cα model for the entire procapsid. (D) The internal surface view of a portion of procapsid density map along an icosahedral threefold axis (labeled “3”), showing the V-shaped densities in red around hexamers (labeled “6”) and pentamers (labeled “5”). The V-shaped densities at pentamers, shown in a black circle, are displayed at a lower contour level than the rest of the map. The density map was low-pass filtered from 3.8 to 7.0 Å, and was radially colored according to the color scheme shown at the bottom except the inset of a pentamer in the black circle using different color scheme shown on the right. (E) Interactions between the coat protein (colored ribbons, N arm colored in blue) and the C-terminal helix-loop-helix motif of the scaffolding protein (red cylinders) in a hexamer. (F) The charge distribution of one procapsid subunit model. The scaffolding protein-binding region (dashed circle) is negatively charged.

Fig. 2.

Asymmetric reconstruction of the P22 procapsid. (A) The asymmetric procapsid map at 8.7-Å resolution. (B) A central slice of the asymmetric reconstruction. The inner two disordered concentric shells (red) are composed mainly of scaffolding proteins. The portal (gray) interacts with the scaffolding proteins at positions pointed by black arrows and with the coat proteins at the positions marked with circles. In (A), the portal density (gray) was segmented out and 12-fold averaged. In both A and B, the density map was color-coded using the same scheme as in Fig. 1D, except the portal is in gray. (C) The C termini of 10 scaffolding proteins are labeled from 1 to 10, from five hexamers (circled) surrounding the portal vertex. The view direction is at the level of, and normal to, the dashed line in B and the portal has been removed for clarity. (D) Side view of the interaction model among the 10 scaffolding protein C termini (red cylinders), coat proteins (ribbons), and the 12-fold averaged portal density (gray). A difference map between the procapsid asymmetric structure and its icosahedrally averaged structure showed no significant difference for the bound scaffolding proteins and capsid shell; the only difference was at the portal. The red cylinders were fit into the scaffolding protein densities of icosahedrally averaged structure.

Fig. 3.

The icosahedral structure of the P22 virion at 4.0-Å resolution and conformational changes upon maturation. (A) A typical cryo-EM image of P22 virion. (B) The Cα backbone model of gp5 (residues 1–425) for the virion. Outside (C) and tangential (D) views of a subunit model from the virion (magenta) and procapsid (cyan) superimposed, showing conformational changes at the long helix, D loop, and E loop. An interior view of a procapsid subunit (E) and of a virion subunit (F). The movement of A-domain tip (orange) and the N arm (navy) are apparent in the virus maturation.

Fig. 4.

Structural difference at the capsomere interface. (A and B) Zoom-in view of local twofold interactions in the procapsid (A) and the virion (B) models.

Fig. 5.

Pathway for capsid assembly, scaffolding protein release and capsid maturation. (A and B) The portal (gray) associates with scaffolding (red) and coat (cyan) proteins to initiate procapsid assembly. The assembly continues with the addition of scaffolding and coat subunits to the growing shell (C) until the full procapsid is assembled (D). DNA is then packaged into the procapsid shell through the channel of the portal by the terminase motor. The scaffolding proteins are released by the electrostatic forces from DNA being packaged and exit through the central large openings of hexamers (E). During the release of scaffolding proteins, conformational changes associated with the maturation transition occur. After capsid expansion and DNA packaging, the tail hub, needle, and tail spikes are attached to the portal to form an infectious virion (magenta) (F). In B and C, the insets show the side views. In D and F, the insets show one hexamer and the adjacent pentamer rotated from the side view to the end-on view.