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

National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2011; 108(4):1355-1360. DOI: 10.1073/pnas.1015739108


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.

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    • "The coat proteins of phages P22 (Chen et al., 2011; Parent et al., 2010a), Sf6 (Parent et al., 2012b), and CUS-3 (Parent et al., 2014) each have an accessory domain inserted (Insertion domain, I-domain) between βF and βG of the β-hinge (magenta in Fig. 1(B) and (F)). Cryo-EM reconstructions revealed that the I-domains are positioned as surface-exposed protrusions from P22, Sf6, and CUS-3 capsids. "
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    ABSTRACT: For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Apr 2015 · Virology
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    • "The contacts between the D-loops and the neighboring subunits are broken during P22 expansion in a manner similar to that seen for the HK97 G-loop–E-loop contacts. This is illustrated in Supplementary Movie S3, where expansion of part of the P22 capsid is modeled (using PDB ID: 2XYY and the P22 mature capsid model PDB ID: 2XYZ) [11] with D-loops colored pink. Mutations that result in the formation T = 4 instead of the usual T = 7 P22 shells map to residue 285 [64],i nah e l i xt h a ti s directly connected to the D-loop in the current P22 models discussed above, suggesting that changes at residue 285 may change capsid size by altering D-loop geometry. "
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    ABSTRACT: The G-loop is a 10-residue glycine-rich loop that protrudes from the surface of the mature bacteriophage HK97 capsid at the C-terminal end of the long backbone helix of major capsid protein subunits. The G-loop is essential for assembly, is conserved in related capsid and encapsulin proteins, and plays its role during HK97 capsid assembly by making crucial contacts between the hill-like hexamers and pentamers in precursor proheads. These contacts are not preserved in the flattened capsomers of the mature capsid. Aspartate 231 in each of the ~ 400 G-loops interacts with lysine 178 of the E-loop (extended loop) of a subunit on an adjacent capsomer. Mutations disrupting this interaction prevented correct assembly and, in some cases, induced abnormal assembly into tubes, or small, incomplete capsids. Assembly remained defective when D231 and K178 were replaced with larger charged residues or when their positions were exchanged. Second-site suppressors of lethal mutants containing substitution D231L replaced the ionic interaction with new interactions between neutral and hydrophobic residues of about the same size: D231L/K178V, D231L/K178I, and D231L/K178N. We conclude that it is not the charge but the size and shape of the side chains of residues 178 and 231 that are important. These two residues control the geometry of contacts between the E-loop and the G-loop, which apparently must be precisely spaced and oriented for correct assembly to occur. We present a model for how the G-loop could control HK97 assembly and identify G-loop-like protrusions in other capsid proteins that may play analogous roles.
    Preview · Article · May 2014 · Journal of Molecular Biology
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    • "The C-terminal helix–turn–helix domain of scaffolding protein is known to interact with coat protein via electrostatic interactions (Cortines et al., 2011; Parent et al., 2005; Parker and Prevelige, 1998). Only parts of scaffolding protein that are directly in contact with the contiguous procapsid are visible in reconstructions, suggesting that scaffolding protein is in varied orientations , likely interacting with other scaffolding protein molecules (Chen et al., 2011; Thuman-Commike et al., 2000). There are several explanations to rectify the difference between biochemical observations and the recent image reconstruction. "
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    ABSTRACT: In vitro assembly of bacteriophage P22 procapsids requires coat protein and sub-stoichiometric concentrations of the internal scaffolding protein. If there is no scaffolding protein, coat protein assembles aberrantly, but only at higher concentrations. Too much scaffolding protein results in partial procapsids. By treating the procapsid as a lattice that can bind and be stabilized by scaffolding protein we dissect procapsid assembly as a function of protein concentration and scaffolding/coat protein ratio. We observe that (i) the coat-coat association is weaker for procapsids than for aberrant polymer formation, (ii) scaffolding protein makes a small but sufficient contribution to stability to favor the procapsid form, and (iii) there are multiple classes of scaffolding protein binding sites. This approach should be applicable to other heterogeneous virus assembly reactions and will facilitate our ability to manipulate such in vitro reactions to probe assembly, and for development of nanoparticles.
    Full-text · Article · Apr 2012 · Virology
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