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A snapshot of viral evolution from genome analysis of the tectiviridae family.

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.

Amino Acid Sequence↗

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements.

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.

Bacillus cereus↗

Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing.

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.

Biotechnology↗

pGIL01, a linear tectiviral plasmid prophage originating from Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 5' extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group.

Amino Acid Sequence↗

Molecular evolution of adenoviruses.

New advances in the field of genetic characterization of adenoviruses originating from different animal species are summarized. Variations seen in the host range and specificity, pathogenicity, genomic arrangement or gene complement are much wider than expected based on previous studies of human adenoviruses. Several exceptional adenoviruses from the two traditional conventional genera are now removed, and proposed to form at least two new genera. The eventual host origin of the new genera, however, is not clarified. Novel results from the genomic and phylogenetic analyses of adenoviruses originating from lower vertebrate species (including reptiles, amphibians and fish) seem to imply that probably five major clusters of adenoviruses exist corresponding to the five major classes of Vertebrata. Adenoviruses, which are now suspected to have common origin with enterobacterium phages from the family Tectiviridae, are perhaps very ancient indeed, and may have undergone a co-evolution with vertebrate hosts.

Adenoviridae↗

Isolation and characterization of Thermus bacteriophages.

One-hundred-fifteen bacteriophage strains were isolated from alkaline hot springs in Iceland, New Zealand, Russia (Kamchatka), and the U.S.A. The phages belonged to the Myoviridae, Siphoviridae, Tectiviridae, and Inoviridae families. Over 50% of isolates were isometric or filamentous. One type of siphovirus had giant tails of over 800 nm in length. Phages were further characterized by host range, genome size, DNA restriction endonuclease digestion patterns, and temperature and pH sensitivity. Myoviruses and tectiviruses had a worldwide distribution. Most phages were narrowly host-specific and all were highly resistant against heating and alkaline and acidic pH. This is the first time that tectiviruses and filamentous phages are reported for bacteria of the Thermus-Deinococcus phylum. The presence of tectiviruses, inoviruses, and myoviruses is attributed to acquisition from ancestral gamma-proteobacteria by horizontal gene transfer.

Alkalies↗

The Bacillus thuringiensis phage GIL01 encodes two enzymes with peptidoglycan hydrolase activity.

Bacteriophage GIL01, infecting Bacillus thuringiensis serovar israelensis, possesses a linear dsDNA genome of 14,931 bp with proteins attached to its 5' extremities and terminal inverted repeats at both ends. Viral particles are sensitive to organic solvents, suggesting that a lipid membrane is present in the capsid. All these characteristics are reminiscent of those found in members of the Tectiviridae family. Sequence analysis of GIL01 revealed the presence of two open reading frames (ORF25 and ORF30) encoding potential lytic enzymes, which were cloned and overexpressed in Escherichia coli. The muralytic activity of these two proteins, designated Mur1 and Mur2 respectively, was confirmed in situ using renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing bacterial cell wall preparations. While Mur2 degrading activity is limited to B. thuringiensis israelensis, Mur1 has a broader cleavage spectrum.

Amino Acid Sequence↗

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.

Adenosine Triphosphatases↗

Preliminary crystallographic analysis of the major capsid protein P2 of the lipid-containing bacteriophage PM2.

PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P2(1)2(1)2, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 A. The crystals diffract to 4 A resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.

Capsid Proteins↗

Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core.

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family TECTIVIRIDAE:

Capsid↗

Comparison of bacteriophages for use in iodine inactivation: batch and continuous flow studies.

Inactivation rates in batch studies for four commonly used surrogate bacteriophages were measured in stable aqueous iodine solutions for the purpose of determining which was the most suited to evaluate iodine disinfection efficacy in batch and continuous flow conditions. Two types of group Leviviridae bacteriophages were used, Type I (MS2) and Type II (GA), along with group Microviridae, Phi-X174, and group Tectiviridae, PRD1. Inactivation was compared at iodine doses of 1.0-1.5 mg l2/l. MS2 was the most susceptible to iodine inactivation of the four phages tested. Inactivation of naked, icosahedral bacteriophages, MS2 and Phi-X174 demonstrated removals to below detection limits (>99.99%) in less than 10 min. Lipid-containing PRD1 and F+ssRNA GA bacteriophages demonstrated the greatest iodine resistance in batch experiments with an average of 1.82 logs of inactivation (98.5%) after 60 min and 1.05 logs of inactivation (91.1%) after 30 min respectively. Similarly, in continuous flow studies through pentaiodide quaternary ammonium strong base resin, MS2, GA and Phi-X174 were more strongly inactivated than PRD1. The lipid component of PRD1 is thought to enhance resistance to iodine over non-lipid-containing bacteriophages by protecting easily oxidized groups on the protein capsid, but further research is needed before proving this hypothesis. The results from this research may provide a surrogate standard for more rigorous and developed research into the mode of iodine disinfection and its inactivation kinetics.

Anion Exchange Resins↗

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.

Adenoviridae↗

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↗

Local average intensity-based method for identifying spherical particles in electron micrographs.

A method is presented that reliably detects spherical viruses from a wide variety of noisy low-contrast electron micrographs. Such detection is one of the first image analysis steps in the computer-aided reconstruction of three-dimensional density distribution models of viruses. Particle detection is based on the comparison of intensity in a circular area and in the surrounding ring followed by a number of tests to validate the potential particles. The only required input from the user in addition to the micrograph is an approximate radius of the particle. The method has been implemented as program ETHAN that has been tested for several different data sets. ETHAN has also successfully been used to detect DNA-less virus particles for an actual reconstruction.

DNA, Viral↗

Probing phage PRD1-specific proteins with monoclonal and polyclonal antibodies.

Four new specificity class MAbs against PRD1 proteins (P6, P7/14, P11, P18) and polyclonal antiserum against the minor capsid protein P5 were produced. The antibodies were used to analyze the phage protein distribution inside the host cell during infection as well as in the virion. The minor component of the capsid, P5, was shown to be located on the surface of the virion. The proteins responsible for particle infectivity were localized to the membrane fraction of the host cells. In addition, by detection with MAbs, genes encoding proteins P14 and P18 were positively localized on the PRD1 genome.

Antibodies, Monoclonal↗

Assembly of membrane-containing bacteriophage PRD1 is dependent on GroEL and GroES.

Assembly of the broad-host-range bacteriophage PRD1 involves translocation of the virus-specific membrane to the inside of the icosahedral protein shell formed of trimeric coat proteins. The formation of PRD1 particles is, in addition to the virus-encoded assembly factors P10 and P17, dependent on GroEL/GroES chaperonins. The chaperonins assist in the folding of the capsid proteins P3 and P5 and in the assembly of viral membrane proteins.

Chaperonin 10↗

Membrane proteins modulate the bilayer curvature in the bacterial virus Bam35.

Biological membranes control the flow of molecules into and out of cells, and they transmit information about the milieu. Structural studies of membrane-containing viruses provide one way to study these membranes in situ. Cryo-electron microscopy and image reconstruction of bacteriophage Bam35 to 7.3 A resolution revealed a membrane bilayer constrained within an icosahedrally symmetric pseudo T = 25 capsid. A total of 60 large transmembrane protein complexes affect the curvature and thickness of the membrane. Here, we describe these membrane parameters quantitatively. Furthermore, we show that Bam35 differs from bacteriophage PRD1 in these parameters, even though the two viruses share the same principles of capsid architecture. Most notably, each virus possesses a tape measure protein suggesting a general mechanism for capsid size determination in icosahedral viruses.

Bacillus thuringiensis↗