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At least 19 recordsLinked to original sources

PubMed1995

DNA packaging orders the membrane of bacteriophage PRD1.

Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat. Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3. The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice. The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane. The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density. The size of the coat does not change upon DNA packaging. Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase. Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses.

Bacteriophages↗

PubMed1994

Expression in Escherichia coli of rat neurotensin receptor fused to membrane proteins from the membrane-containing bacteriophage PRD1.

Bacteriophage PRD1 is a membrane-containing phage which could be used for expression of foreign membrane proteins such as neurotensin receptor (NTR), a seven-helix G-protein coupled receptor. To ensure recognition of NTR by the phage system six different fusion genes were constructed, each encoding a different phage integral membrane protein fused to the N-terminus of NTR, and expression of the fusion proteins in Escherichia coli was analysed. Here we report the identification of two fusion constructs that retained the function of NTR in E. coli. This provides the basis to develop the phage system as a heterologous expression system for seven-helix receptors.

Amino Acid Sequence↗

PubMed2000

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.

Capsid↗

PubMed2002

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.

Bacteriophage PRD1↗

PubMed1989

The organization of the right-end early region of bacteriophage PRD1 genome.

Bacteriophage PRD1 is the only protein-primed DNA replication system known to operate in Escherichia coli. The left-genome end of PRD1 contains the early genes for the terminal protein and the DNA polymerase. These genes have been sequenced and the proteins have been produced separately. In this investigation we completed the analysis of the PRD1 early DNA regions by cloning and sequencing the right end genome containing early genes XII and XIX. We compared the structure of the right- and left-terminal regions. The genome organization of both ends was found to be rather uniform. The inverted terminal repeats, the first promoters and the first translation start codons are located almost exactly at the same distance from the genome ends. The PRD1 early gene products, P12 and P19, do not share similarities with proteins found in other protein-primed replication systems.

Amino Acid Sequence↗

PubMed2001

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.

Bacteriophage PRD1↗

PubMed2001

A minor capsid protein P30 is essential for bacteriophage PRD1 capsid assembly.

Bacteriophage PRD1 is a double-stranded DNA virus infecting Gram-negative hosts. It has a membrane component located in the interior of the isometric capsid. In addition to the major capsid protein P3, the capsid contains a 9 kDa protein P30. Protein P30 is proposed to be located between the adjacent facets of the icosahedral capsid and is required for stable capsid assembly. In its absence, an empty phage-specific membrane vesicle is formed. The major protein component of this vesicle is a phage-encoded assembly factor, protein P10, that is not present in the final structure.

Bacteriophage PRD1↗

PubMed1983

Molecular cloning of bacteriophage PRD1 genomic fragments.

DNA from bacteriophage PRD1 was extracted and partially digested with restriction endonuclease HaeII. The digest was cloned into the PstI site of plasmid pBR322 by homopolymer tailing with guanidylate tails on the plasmid and cytidylate tails on the phage DNA. Insert bearing plasmids were isolated by transforming E. coli strains for tetracycline resistance and screening for ampicillin sensitivity. These strains were then screened for the ability to accomplish marker rescue of nonsense mutants of bacteriophage PRD1. Additional clones were isolated by screening transformants with radioactively labeled probe PRD1 DNA fragments using colony hybridization. A genetic map was generated by the marker rescue capabilities of overlapping cloned inserts. This map allowed the ordering of fourteen of the known PRD1 complementation groups.

Bacteriophages↗

PubMed1991

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.

Bacteriophages↗

PubMed2002

The lytic enzyme of bacteriophage PRD1 is associated with the viral membrane.

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.

Bacteriolysis↗

PubMed1984

Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome.

DNA of bacteriophage PRD1 has protein P8 at its termini. Extracts of infected cells are able to derivatize P8 in vitro with labeled dGTP. Two early proteins, P1 and P8, products of genes I and VIII, respectively, are the only phage proteins necessary for the formation of the protein P8-dGMP complex. This was shown by complementation of extracts from cells infected with mutants and by use of extracts from cells carrying cloned genes I and VIII. With Escherichia coli mutants that are temperature sensitive for DNA synthesis, it was possible to show that the formation of the protein P8-dGMP complex was dependent upon the host replication apparatus. The analysis of the purified protein P8-dGMP complex by hydrolysis and enzymatic digestion showed that there is a covalent phosphodiester bond between tyrosine and 5'-dGMP.

Bacteria↗

PubMed2007

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.

Bacteriophage PRD1↗

PubMed2003

Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope.

Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.

Amino Acid Sequence↗

PubMed1983

Identification of a protein bound to the termini of bacteriophage PRD1 DNA.

Lipid-containing bacteriophage PRD1 has a double-stranded DNA genome of about 14,500 nucleotide base pairs. The phage can infect Escherichia coli and Salmonella typhimurium as well as other gram-negative bacteria harboring an appropriate plasmid. [35S]methionine label is incorporated into the DNA band early in infection. The label remains associated with DNA through phenol extraction and boiling with sodium dodecyl sulfate. Nuclease treatment of the genome released a protein which migrated as an early phage-specific protein (P8). This protein is also necessary for phage DNA replication. By restriction enzyme analysis it was shown that protein was associated with the terminal restriction fragments. Extracts of infected cells catalyzed the labeling of protein P8 with [alpha-32P]dGTP.

Bacteriophages↗

PubMed1993

Protein-primed DNA replication: role of inverted terminal repeats in the Escherichia coli bacteriophage PRD1 life cycle.

Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybrid phages made in vitro verified this observation. However, genomes in which the dissimilar DNA termini had regained identical sequences were also detected. These observations are explained by a DNA replication model involving two not mutually exclusive pathways. The generality of this model in protein-primed DNA replication is discussed.

Amino Acid Sequence↗

PubMed2002

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry.

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.

Amino Acid Sequence↗

PubMed1993

Overproduction, purification, and characterization of DNA-binding protein P19 of bacteriophage PRD1.

The early protein, P19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5 alpha cells. The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity. Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios. P19 was capable of fully protecting ssDNA against nuclease P1. Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P19 binding. The results suggest that P19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P19 multimers.

Bacteriophages↗

PubMed1982

Isolation of nonsense mutants of lipid-containing bacteriophage PRD1.

We isolated nonsense mutants of bacteriophage PRD1, a lipid-containing polyhedral virus capable of infecting many genera of gram-negative bacteria. These mutants were grouped into 19 classes on the basis of genetic complementation and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. PRD1 infection led to the synthesis of at least 25 viral proteins, 17 of which were components of mature virions. The synthesis of proteins fell into the following three classes: very early, middle early, and late. Two of the very early proteins, P1 and P8, had an effect on DNA synthesis, host protein synthesis shutoff, and the turning on of middle and late protein synthesis. Another very early protein, P12, was involved in the shutoff of early protein synthesis. Two genes were identified as affecting lysis of the host. One appeared to be a lysin, whereas the other was an accessory lytic factor.

Genetic Complementation Test↗