Procapsid Assembly Research Articles

Cryo-electron microscopy structures of capsids and in situ portals of DNA-devoid capsids of human cytomegalovirus

The portal-scaffold complex is believed to nucleate the assembly of herpesvirus procapsids. During capsid maturation, two events occur: scaffold expulsion and DNA incorporation. The portal-scaffold interaction and the conformational changes that occur to the portal during the different stages of capsid formation have yet to be elucidated structurally. Here we present high-resolution structures of the A- and B-capsids and in-situ portals of human cytomegalovirus. We show that scaffolds bind to the hydrophobic cavities formed by the dimerization and Johnson-fold domains of the major capsid proteins. We further show that 12 loop-helix-loop fragments—presumably from the scaffold domain—insert into the hydrophobic pocket of the portal crown domain. The portal also undergoes significant changes both positionally and conformationally as it accompanies DNA packaging. These findings unravel the mechanism by which the portal interacts with the scaffold to nucleate capsid assembly and further our understanding of scaffold expulsion and DNA incorporation.

Assembly and Capsid Expansion Mechanism of Bacteriophage P22 Revealed by High-Resolution Cryo-EM Structures.

The formation of many double-stranded DNA viruses, such as herpesviruses and bacteriophages, begins with the scaffolding-protein-mediated assembly of the procapsid. Subsequently, the procapsid undergoes extensive structural rearrangement and expansion to become the mature capsid. Bacteriophage P22 is an established model system used to study virus maturation. Here, we report the cryo-electron microscopy structures of procapsid, empty procapsid, empty mature capsid, and mature capsid of phage P22 at resolutions of 2.6 Å, 3.9 Å, 2.8 Å, and 3.0 Å, respectively. The structure of the procapsid allowed us to build an accurate model of the coat protein gp5 and the C-terminal region of the scaffolding protein gp8. In addition, interactions among the gp5 subunits responsible for procapsid assembly and stabilization were identified. Two C-terminal α-helices of gp8 were observed to interact with the coat protein in the procapsid. The amino acid interactions between gp5 and gp8 in the procapsid were consistent with the results of previous biochemical studies involving mutant proteins. Our structures reveal hydrogen bonds and salt bridges between the gp5 subunits in the procapsid and the conformational changes of the gp5 domains involved in the closure of the local sixfold opening and a thinner capsid shell during capsid maturation.

Atomic structure of the Epstein-Barr virus portal

Herpesviridae is a vast family of enveloped DNA viruses that includes eight distinct human pathogens, responsible for diseases that range from almost asymptomatic to severe and life-threatening. Epstein-Barr virus infects B-cells and epithelial cells, causing infectious mononucleosis, as well as a number of cancers. Epstein-Barr infection cannot be cured since neither vaccine nor antiviral drug treatments are available. All herpesviruses contain a linear double-stranded DNA genome, enclosed within an icosahedral capsid. Viral portal protein plays a key role in the procapsid assembly and DNA packaging. The portal is the entrance and exit pore for the viral genome, making it an attractive pharmacological target for the development of new antivirals. Here we present the atomic structure of the portal protein of Epstein-Barr virus, solved by cryo-electron microscopy at 3.5 Å resolution. The detailed architecture of this protein suggests that it plays a functional role in DNA retention during packaging.

Architect of Virus Assembly: the Portal Protein Nucleates Procapsid Assembly in Bacteriophage P22.

Tailed double-stranded DNA (dsDNA) bacteriophages, herpesviruses, and adenoviruses package their genetic material into a precursor capsid through a dodecameric ring complex called the portal protein, which is located at a unique 5-fold vertex. In several phages and viruses, including T4, Φ29, and herpes simplex virus 1 (HSV-1), the portal forms a nucleation complex with scaffolding proteins (SPs) to initiate procapsid (PC) assembly, thereby ensuring incorporation of only one portal ring per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We have developed an in vitro P22 assembly assay where portal protein is coassembled into procapsid-like particles (PLPs). Scaffolding protein also catalyzes oligomerization of monomeric portal protein into dodecameric rings, possibly forming a scaffolding protein-portal protein nucleation complex that results in one portal ring per P22 procapsid. Here, we present evidence substantiating that the P22 portal protein, similarly to those of other dsDNA viruses, can act as an assembly nucleator. The presence of the P22 portal protein is shown to increase the rate of particle assembly and contribute to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature that is likely conserved among these classes of dsDNA viruses.IMPORTANCE The existence of a single portal ring is essential to the formation of infectious virions in the tailed double-stranded DNA (dsDNA) phages, herpesviruses, and adenoviruses and, as such, is a viable antiviral therapeutic target. How only one portal is selectively incorporated at a unique vertex is unclear. In many dsDNA viruses and phages, the portal protein acts as an assembly nucleator. However, early work on phage P22 assembly in vivo indicated that the portal protein did not function as a nucleator for procapsid (PC) assembly, leading to the suggestion that P22 uses a unique mechanism for portal incorporation. Here, we show that portal protein nucleates assembly of P22 procapsid-like particles (PLPs). Addition of portal rings to an assembly reaction increases the rate of formation and yield of particles and corrects improper particle morphology. Our data suggest that procapsid assembly may universally initiate with a nucleation complex composed minimally of portal and scaffolding proteins (SPs).

A viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly.

Most double-stranded DNA viruses package genetic material into empty precursor capsids (or procapsids) through a dodecameric portal protein complex that occupies 1 of the 12 vertices of the icosahedral lattice. Inhibiting incorporation of the portal complex prevents the formation of infectious virions, making this step an excellent target for antiviral drugs. The mechanism by which a sole portal assembly is selectively incorporated at the special vertex is unclear. We recently showed that, as part of the DNA packaging process for bacteriophage P22, the dodecameric procapsid portal changes conformation to a mature virion state. We report that preformed dodecameric rings of P22 portal protein, as opposed to portal monomers, incorporate into nascent procapsids, with preference for the procapsid portal conformation. Finally, a novel role for P22 scaffolding protein in triggering portal ring formation from portal monomers is elucidated and validated by incorporating de novo assembled portal rings into procapsids.

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the β-barrel fold. A hydrogen-bonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitive-folding mutation. The pKa of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ∼4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo By contrast, the mutants have only minor effects on capsid expansion or stability in vitro The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly.

Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES.

The putative herpes simplex virus 1 chaperone protein UL32 modulates disulfide bond formation during infection.

During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation. Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.

The role of the coat protein A-domain in p22 bacteriophage maturation.

Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.

High-resolution structure of a virally encoded DNA-translocating conduit and the mechanism of DNA penetration.

Although ϕX174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the host cell surface. Reminiscent of a double-stranded DNA phage tail in form and function, the H tube transports the single-stranded ϕX174 genome across the Escherichia coli cell wall. The 2.4-Å resolution H-tube crystal structure suggests functional and energetic mechanisms that may be common features of DNA transport through virally encoded conduits.

The delta domain of the HK97 major capsid protein is essential for assembly

The 102 residue N-terminal extension of the HK97 major capsid protein, the delta domain, is normally present during the assembly of immature HK97 procapsids, but it is removed during maturation like well-known internal scaffolding proteins of other tailed phages and herpesviruses. The delta domain also shares other unusual properties usually found in other viral and phage scaffolding proteins, including its location on the inside of the capsid, a high predicted and measured α-helical content, and an additional prediction for the ability to form parallel coiled-coils. Viral scaffolding proteins are essential for capsid assembly and phage viability, so we tested whether the HK97 delta domain was essential for capsid assembly. We studied the effects of deleting all or parts of the delta domain on capsid assembly and on complementation of capsid-protein-defective phage, and our results demonstrate that the delta domain is required for HK97 capsid assembly.

Electrostatic Interactions Drive the Self-Assembly and the Transcription Activity of the Pseudomonas Phage ϕ6 Procapsid

Assembly of an empty procapsid is a crucial step in the formation of many complex viruses. Here, we used the self-assembly system of the double-stranded RNA bacteriophage ϕ6 to study the role of electrostatic interactions in a scaffolding-independent procapsid assembly pathway. We demonstrate that ϕ6 procapsid assembly is sensitive to salt at both the nucleation and postnucleation steps. Furthermore, we observed that the salt sensitivity of ϕ6 procapsid-directed transcription is reversible.

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

An Intramolecular Chaperone Inserted in Bacteriophage P22 Coat Protein Mediates Its Chaperonin-independent Folding

The bacteriophage P22 coat protein has the common HK97-like fold but with a genetically inserted domain (I-domain). The role of the I-domain, positioned at the outermost surface of the capsid, is unknown. We hypothesize that the I-domain may act as an intramolecular chaperone because the coat protein folds independently, and many folding mutants are localized to the I-domain. The function of the I-domain was investigated by generating the coat protein core without its I-domain and the isolated I-domain. The core coat protein shows a pronounced folding defect. The isolated I-domain folds autonomously and has a high thermodynamic stability and fast folding kinetics in the presence of a peptidyl prolyl isomerase. Thus, the I-domain provides thermodynamic stability to the full-length coat protein so that it can fold reasonably efficiently while still allowing the HK97-like core to retain the flexibility required for conformational switching during procapsid assembly and maturation.

The energetic contributions of scaffolding and coat proteins to the assembly of bacteriophage procapsids

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.

The Bacteriophage Lambda gpNu3 Scaffolding Protein Is an Intrinsically Disordered and Biologically Functional Procapsid Assembly Catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition ( T m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer–dimer equilibrium ( K d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.

Conformational Changes in Bacteriophage P22 Scaffolding Protein Induced by Interaction with Coat Protein

Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60–300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini.

Peering Down the Barrel of a Bacteriophage Portal: The Genome Packaging and Release Valve in P22

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia etal. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins.

Intracellular Assembly of Cyanophage Syn5 Proceeds through a Scaffold-Containing Procapsid

Syn5 is a marine cyanophage that is propagated on the marine photosynthetic cyanobacterial strain Synechococcus sp. WH8109 under laboratory conditions. Cryoelectron images of this double-stranded DNA (dsDNA) phage reveal an icosahedral capsid with short tail appendages and a single novel hornlike structure at the vertex opposite the tail. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. The one-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ∼45 min, a latent phase of ∼60 min, and a burst size of 20 to 30 particles per cell at 28°C. SDS-PAGE and Western blot analysis of cell lysates at different times after infection showed the synthesis of major virion proteins and their increase as the infection progressed. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. It migrated anomalously on SDS-PAGE, similar to the phage T7 scaffolding protein. Particles lacking DNA but containing the coat and scaffolding proteins were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA but filled with protein density, presumably scaffolding protein. These findings suggest that the cyanophages form infectious virions through the initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid-mediated assembly pathway may have originated with the cyanophages.