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 ¼ 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.sDNA viruses infecting both prokaryotes and eukaryotes share a common assembly pathway proceeding from a precursor (procapsid) to an infectious virion (1-4). In addition to the coat proteins, the procapsid requires scaffolding proteins, absent from the virion, for proper assembly, and a portal for DNA packaging and subsequent DNA ejection. However, despite a half-century of research on icosahedral viruses, it remains unclear how initially identical subunits adopt both hexameric and pentameric conformations in the virus and select the correct locations needed to form closed shells of the proper size (5). Packaging of DNA through the portal is accompanied by the exit of scaffolding proteins from the procapsid and conformational changes in the coat proteins as the capsid matures (2, 6).Understanding the molecular mechanisms of dsDNA virus assembly and maturation requires knowledge of the interactions among the coat, scaffolding, and portal proteins, all of which are essential for these processes. X-ray crystallography (7-9) and electron cryomicroscopy (cryo-EM) (10-12) have yielded nearatomic to atomic resolution models of several dsDNA icosahedral viruses and provided a structural framework of interactions among their coat proteins. However, the structural details of procapsid portal incorporation, scaffolding protein bind...

Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry

“…The other additional density, located at the C1 capsid quasi-2-fold, bridges between neighboring hexameric capsomers and between neighboring hexameric and pentameric capsomers, thus aiding capsid stability. Similar densities at the capsomer interfaces had been observed in phages BPP-1, P-SSP7, and P22 (17,18,36). In Bordetella phage BPP-1 these densities were attributed to a cement protein.…”

“…In the HK97 structure, these regions have the largest structural deviation among the seven subunits that form an asymmetric unit of the HK97 capsid (27). Also, these portions of the HK97 fold were shown to have the most differences among different phages (12,14,18,36). The number of residues in gp16 of C1 is comparable to that of the HK97 capsid protein (392 a.a. versus 385 a.a.).…”

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7

“…S2B), as shown before in the procapsid structures of T7 (19,26) and other tailed dsDNA phages (10,27). In the icosahedral reconstruction, all of the nonicosahedral features, including the scaffolding protein (gp9) inside the gp10 shell and the portal/core stack complex, are smeared.…”

“…Symmetry mismatches also occur in the capping and branching of actin filaments to control cell shapes (7), and the reciprocating conformational changes of the two barrels of GroEL (Ping-Pong mode) to fold protein substrates (8). Although the details of symmetric structures have been routinely visualized, the details of both asymmetry and symmetry mismatch have only recently become visualizable with the development of single-particle cryo-EM and associated asymmetric 3D reconstruction methods (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Both the symmetry-mismatched, coaxial 12-fold portal ring and the 5-fold capsid of several bacteriophages (9-11, 13, 15-18) were resolved simultaneously in asymmetric reconstructions, i.e., reconstructions in which no symmetry was imposed.…”

Atomic Structure of Bacteriophage Sf6 Tail Needle Knob

“…This contributes to the slightly tighter packing of the Sf6 tail needle knob relative to the PRD1/adenovirus knobs, which is also exemplified by its slightly narrower external width (45,47, and 60 Å for Sf6, PRD1, and adenovirus knobs, respectively) (Fig. 4, A-C).…”