Viral capsid assembly and stability in tailed, dsDNA phage and Herpesviridae are achieved by various means including chemical cross-links (unique to HK97), or auxiliary proteins (lambda, T4, ϕ29, and herpesviruses). All these viruses have coat proteins (CP) with a conserved, HK97-like core structure. We used a combination of trypsin digestion, gold-labeling, cryo-electron microscopy, 3D image reconstruction, and comparative modeling to derive two independent, pseudo-atomic models of bacteriophage P22 CP: before and after maturation. P22 capsid stabilization results from inter-subunit interactions among N-terminal helices and an extensive “P-loop”, which obviate the need for cross-links or auxiliary proteins. P22 CP also has a telokin-like, Ig domain that stabilizes the monomer fold so that assembly may proceed via individual subunit addition rather than via preformed capsomers as occurs in HK97. Hence, the P22 CP structure may be a paradigm for understanding how monomers assemble in viruses like ϕ29 and HSV-1.
Bacteriophage assembly frequently begins with the formation of a precursor capsid that serves as a DNA packaging machine. The DNA packaging is accompanied by a morphogenesis of the small round precursor capsid into a large polyhedral DNA-containing mature phage. In vitro, this transformation can be induced by heat or chemical treatment of P22 procapsids. In this work, we examine bacteriophage P22 morphogenesis by comparing three-dimensional structures of capsids expanded both in vitro by heat treatment and in vivo by DNA packaging. The heat-expanded capsid reveals a structure that is virtually the same as the in vivo expanded capsid except that the pentons, normally present at the icosahedral fivefold positions, have been released. The similarities of these two capsid structures suggest that the mechanism of heat expansion is similar to in vivo expansion. The loss of the pentons further suggests the necessity of specific penton-hexon interactions during expansion. We propose a model whereby the penton-hexon interactions are stabilized through interactions of DNA, coat protein, and other minor proteins. When considered in the context of other studies using chemical or heat treatment of capsids, our study indicates that penton release may be a common trend among double-stranded DNA containing viruses.
The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980; Newcomb et al., 1999). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine (Earnshaw, Hendrix, and King, 1980; Heymann et al., 2003). DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.
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.
Some capsid proteins built on the ubiquitous HK97-fold have accessory domains that impart specific functions. Bacteriophage P22 coat protein has a unique inserted I-domain. Two prior I-domain models from sub-nanometer cryoEM reconstructions differed substantially. Therefore, the NMR structure of the I-domain was determined, which also was used to improve cryoEM models of coat protein. The I-domain has an anti-parallel 6-stranded β-barrel fold, previously not observed in HK97-fold accessory domains. The D-loop, which is dynamic both in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. A newly described S-loop is important for capsid size determination, likely through intra-subunit interactions. Ten of eighteen coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.
Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein.
A stepwise addition protocol was developed to display cargo using bacteriophage P22 capsids and the phage decorator (Dec) protein. Three-dimensional image reconstructions of frozen-hydrated samples of P22 particles with nanogold-labeled Dec bound to them revealed the locations of the N- and C- termini of Dec. Each terminus is readily accessible for molecular display through affinity tags such as nickel-nitrilotriacetic acid, providing a total of 240 cargo-binding sites. Dec was shown by circular dichroism to be a β–sheet rich protein, and fluorescence anisotropy binding experiments demonstrated that Dec binds to P22 heads with high (~110 nM) affinity. Dec also binds to P22 nanotubes, which are helically symmetric assemblies that form when the P22 coat protein contains the F170A amino acid substitution. Several classes of tubes with Dec bound to them were visualized by cryo-electron microscopy and their three-dimensional structures were determined by helical reconstruction methods. In all instances, Dec trimers bound to P22 capsids and nanotubes at positions where three neighboring capsomers (oligomers of six coat protein subunits) lie in close proximity to one another. Stable interactions between Dec and P22 allow for the development of robust, nanoscale size, display vehicles.
Icosahedral capsid assembly is an example of a reaction controlled solely by the interactions of the proteins involved. Bacteriophage P22 procapsids can be assembled in vitro by mixing coat and scaffolding proteins in a nucleation-limited reaction, where scaffolding protein directs the proper assembly of coat protein. Here, we investigated the effect of the buffer composition on the interactions necessary for capsid assembly. Different concentrations of various salts, chosen to follow the electroselectivity series for anions, were added to the assembly reaction. The concentration and type of salt was found to be crucial for proper nucleation of procapsids. Nucleation in low salt concentrations readily occurred but led to bowl-like partial procapsids, as visualized by negative stain electron microscopy. The edge of the partial capsids remained assembly-competent since coat protein addition triggered procapsid completion. The addition of salt to the partial capsids also caused procapsid completion. In addition, each salt affected both assembly rates and the extent of procapsid formation. We hypothesize that low salt conditions increase the coat protein:scaffolding protein affinity, causing excessive nuclei to form, which decreases coat protein levels leading to incomplete assembly.
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