Integral membrane protein P16 of bacteriophage PRD1 stabilizes the adsorption vertex structure

The icosahedral membrane-containing double-stranded DNA bacteriophage PRD1 has a labile receptor binding spike complex at the vertices. This complex, which is analogous to that of adenovirus, is formed of the penton protein P31, the spike protein P5, and the receptor binding protein P2. Upon infection, the internal phage membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection. We describe here a new class of PRD1 mutants lacking virion-associated integral membrane protein P16. P16 links the spike complex to the viral membrane and is necessary for spike stability. We also show that the unique vertex used for DNA packaging is intact in the P16-deficient particle, indicating that the 11 adsorption vertices and the 1 portal vertex are functionally and structurally distinct.     

Bacteriophage PRD1 contains a labile receptor-binding structure at each vertex.

Bacteriophage PRD1 is a membrane-containing virus with an unexpected similarity to adenovirus. We mutagenized unassigned PRD1 genes to identify minor capsid proteins that could be structural or functional analogs to adenovirus proteins.We report here the identification of an amber mutant, sus525, in an essential PRD1 gene XXXI. The gene was cloned and the gene product was overexpressed and purified to near homogeneity. Analytical ultracentrifugation and gel filtration showed that P31 is a homopentamer of about 70 kDa. The protein was shown to be accessible on the virion surface and its absence in the sus525 particles led to the deficiency of two other viral coat proteins, protein P5 and the adsorption protein P2. Cryo-electron microscopy and image reconstruction of the sus525 particles indicate that these proteins are located on the capsid vertices, because in these particles the entire vertex structure was missing along with the peripentonal major capsid protein P3 trimers. Sus525 particles package DNA effectively but loose it upon purification.All of the PRD1 vertex structures are labile and potentially capable of mediating DNA delivery; this is in contrast to other dsDNA phages which employ a single vertex for packaging and delivery. We propose that this arises from a symmetry mismatch between protein P2 and the pentameric P31 in analogy to that between the adenovirus penton base and the receptor-binding spike.     

Solution structure of bacteriophage PRD1 vertex complex

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.     

The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture

Comparisons of bacteriophage PRD1 and adenovirus protein structures and virion architectures have been instrumental in unraveling an evolutionary relationship and have led to a proposal of a phylogeny-based virus classification. The structure of the PRD1 spike protein P5 provides further insight into the evolution of viral proteins. The crystallized P5 fragment comprises two structural domains: a globular knob and a fibrous shaft. The head folds into a ten-stranded jelly roll beta barrel, which is structurally related to the tumor necrosis factor (TNF) and the PRD1 coat protein domains. The shaft domain is a structural counterpart to the adenovirus spike shaft. The structural relationships between PRD1, TNF, and adenovirus proteins suggest that the vertex proteins may have originated from an ancestral TNF-like jelly roll coat protein via a combination of gene duplication and deletion.     

Minor proteins,mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.     

The receptor binding protein P2 of PRD1, a virus targeting antibiotic-resistant bacteria,has a novel fold suggesting multiple functions

Bacteriophage PRD1 is unusual, with an internal lipid membrane, but has striking resemblances to adenovirus that include receptor binding spikes. The PRD1 vertex complex contains P2, a 590 residue monomer that binds to receptors on antibiotic-resistant strains of E. coli and so is the functional counterpart to adenovirus fiber. P2 structures from two crystal forms, at 2.2 and 2.4 A resolution, reveal an elongated club-shaped molecule with a novel beta propeller "head" showing pseudo-6-fold symmetry. An extended loop with another novel fold forms a long "tail" containing a protruding proline-rich "fin." The head and fin structures are well suited to recognition and attachment, and the tail is likely to trigger the processes of vertex disassembly, membrane tube formation, and subsequent DNA injection.     

The tailless icosahedral membrane virus PRD1 localizes the proteins involved in genome packaging and injection at a unique vertex

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.     

Stable Packaging of Phage PRD1 DNA Requires Adsorption Protein P2, Which Binds to the IncP Plasmid-Encoded Conjugative Transfer Complex

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent K(d) of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.     

Efficient DNA packaging of bacteriophage PRD1 requires the unique vertex protein P6

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.     

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.     

A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.     

The unique vertex of bacterial virus PRD1 is connected to the viral internal membrane

Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex. Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane. The packaging of PRD1 is known to proceed via preformed empty capsids. Now, a unique vertex has been shown to exist in PRD1. We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22. These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle.     

Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5′-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1

Bacteriophage PRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid. Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber. P2 has been crystallized by the "hanging-drop" method of vapor diffusion and two different crystal forms were obtained. Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals. These grew to a size of 0.08 x 0.08 x 0.50 mm(3) and diffracted to 2.6 A resolution. They have the orthorhombic space group P222(1), with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A. A few single crystals of a second form were grown without seeding under slightly different conditions. A parallelepiped crystal (0.10 x 0.10 x 0.35 mm(3)), with space group C222(1) and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution. A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis. The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

Assembly of bacteriophage PRD1 spike complex: role of the multidomain protein P5

The spike structure of bacteriophage PRD1 is comprised of proteins P2, P5, and P31. It resembles the corresponding receptor-binding structure of adenoviruses. We show that purified recombinant protein P5 is an elongated (30 x 2.7 nm; R(h) = 5.5 nm), multidomain trimer which can slowly associate into nonamers. Cleavage of the 340 amino acid long P5 with collagenase yields 2 fragments. The larger, 205 amino acid long C-terminal fragment appears to contain the residues responsible for the trimerization of the protein, whereas the smaller N-terminal part mediates the interaction of P5 with the pentameric vertex protein P31 (24 x 2.5 nm, R(h) = 4.2 nm). In addition, the presence of the N-terminal sequence is required for the formation of the P5 nonamer. The results presented here suggest that P5 and P31 form an elongated adaptor complex at the 5-fold vertexes of the virion which anchors the adsorption protein P2 (21 x 2.5 nm; R(h) = 4.1 nm). Our results also suggest that the P5 trimer forms a substantial part of the viral spike shaft that was previously thought to be composed exclusively of protein P2.     

Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.     

Structural study of the lipid-containing bacteriophage PRD1 and its capsid and DNA components by laser Raman spectroscopy

The Raman spectrum of a virus contains the structural signature of each of its molecular components (Thomas, 1987). We report the first Raman spectrum obtained from an intact, lipid-containing virus--the icosahedral bacteriophage PRD1--and show that this spectrum contains characteristic structure markers for the major capsid protein, the packaged double-stranded DNA genome, and the viral membrane which resides between the capsid and DNA. We find that the packaged genome of PRD1 exhibits Raman markers typical of the B-DNA secondary structure. Comparison of the Raman spectrum of the packaged DNA with that of protein-free DNA extracted from the virion shows further that the B-form secondary structure is not significantly perturbed by packaging in the virion. The Raman signature of the PRD1 membrane, monitored within the virion at 4 degrees C, is that of a phospholipid liquid-crystalline phase. The PRD1 capsid, which comprises several hundred copies of the major coat protein P3 (product of viral gene III) and a few copies of minor proteins, incorporates P3 capsomers predominantly in the beta-sheet conformation. The beta-sheet structure of P3 is maintained in the fully assembled PRD1 virion, as well as in the empty capsid. The present results demonstrate the feasibility of obtaining structural information from the three different classes of biomolecules--nucleic acid, protein, and lipid--which constitute a membrane-lined virus particle. Our results also demonstrate that the coat protein and double-stranded DNA components of a lipid-containing bacteriophage share many structural features in common with bacteriophage lacking a lipid membrane.     

Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.

DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.