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At least 19 recordsLinked to original sources
PubMed2006

Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses.

Single-stranded RNA (ssRNA) bacteriophages of the family Leviviridae infect gram-negative bacteria. They are restricted to a single host genus. Phage PRR1 is an exception, having a broad host range due to the promiscuity of the receptor encoded by the IncP plasmid. Here we report the complete genome sequence of PRR1. Three proteins homologous with those of other ssRNA phages, i.e., maturation, coat, and replicase proteins, were identified. A fourth protein has a lysis function. Comparison of PRR1 with other members of the Leviviridae family places PRR1 in the genus Levivirus with some characteristics more similar to those of members of the genus Allolevivirus.

Allolevivirus↗
PubMed1997

The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses.

The three-dimensional structure of the small T=3 RNA bacteriophage GA has been determined at 3.4 A resolution. The structure was solved by molecular replacement, using the phage MS2 as an initial model. A comparison of the protein shells of the four related phages GA, MS2, fr and Qbeta was carried out in order to define structural features of particular importance for their assembly and specific RNA interaction. A high degree of similarity was found in the RNA binding sites, whereas larger structural differences are located in the loop regions of the coat proteins, especially in the FG loops forming 5-fold and quasi-6-fold contacts. The overall arrangement of the protein subunits in the shells of these phages is very similar, although the details of the interactions differ. The few conserved interactions are suggested to govern the subunit packing during assembly.

Amino Acid Sequence↗
PubMed2002

Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages.

The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q(beta), which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q(beta) suggests that, in an evolutionary sense, AP205 should be placed between Q(beta) and MS2. A phylogenetic tree for the ssRNA phages is presented.

3' Untranslated Regions↗
PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗
PubMed2004

Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

Animals↗
PubMed2001

Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophage (family Leviviridae).

Bacteriophage of the family Leviviridae have played an important role in molecular biology where representative species, such as Q beta and MS2, have been studied as model systems for replication, translation, and the role of secondary structure in gene regulation. Using nucleotide sequences from the coat and replicase genes we present the first statistical estimate of phylogeny for the family Leviviridae using maximum-likelihood and Bayesian estimation. Our analyses reveal that the coliphage species are a monophyletic group consisting of two clades representing the genera Levivirus and Allolevivirus. The Pseudomonas species PP7 diverged from its common ancestor with the coliphage prior to the ancient split between these genera and their subsequent diversification. Differences in genome size, gene composition, and gene expression are shown with a high probability to have changed along the lineage leading to the Allolevivirus through gene expansion. The change in genome size of the Allolevivirus ancestor may have catalyzed subsequent changes that led to their current genome organization and gene expression.

Allolevivirus↗
PubMed2005

Multiple sequence alignments of partially coding nucleic acid sequences.

BACKGROUND: High quality sequence alignments of RNA and DNA sequences are an important prerequisite for the comparative analysis of genomic sequence data. Nucleic acid sequences, however, exhibit a much larger sequence heterogeneity compared to their encoded protein sequences due to the redundancy of the genetic code. It is desirable, therefore, to make use of the amino acid sequence when aligning coding nucleic acid sequences. In many cases, however, only a part of the sequence of interest is translated. On the other hand, overlapping reading frames may encode multiple alternative proteins, possibly with intermittent non-coding parts. Examples are, in particular, RNA virus genomes. RESULTS: The standard scoring scheme for nucleic acid alignments can be extended to incorporate simultaneously information on translation products in one or more reading frames. Here we present a multiple alignment tool, codaln, that implements a combined nucleic acid plus amino acid scoring model for pairwise and progressive multiple alignments that allows arbitrary weighting for almost all scoring parameters. Resource requirements of codaln are comparable with those of standard tools such as ClustalW. CONCLUSION: We demonstrate the applicability of codaln to various biologically relevant types of sequences (bacteriophage Levivirus and Vertebrate Hox clusters) and show that the combination of nucleic acid and amino acid sequence information leads to improved alignments. These, in turn, increase the performance of analysis tools that depend strictly on good input alignments such as methods for detecting conserved RNA secondary structure elements.

Algorithms↗
PubMed1995

Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.

We report the complete nucleotide sequence of the single-stranded RNA phage PP7 from Pseudomonas aeruginosa. There are three open reading frames which code for apparent protein homologues of the single-stranded RNA coliphages, i.e., maturation protein, coat protein, and replicase. A fourth overlapping reading frame exists that probably encodes a lysis protein, similar to what has been found in the group A coliphages such as MS2. The genetic map of PP7 is colinear with group A coliphages and we accordingly classify the phage as a levivirus. There is, generally speaking, no significant nucleotide sequence identity between PP7 and the coliphages except for a few regions where homologous parts of proteins are encoded, most notable in the replicase gene. In these regions the nucleotide sequence similarity between PP7 and MS2 is no greater than between PP7 and the group B coliphages such as Q beta. Surprisingly, Q beta and MS2 are no closer to each other than they are to PP7. Several regulatory RNA secondary structure features that are present in the coliphages were identified also in PP7 RNA although the sequences involved cannot be aligned. Among these are the coat protein binding helix at the start of the replicase gene, structures at the 5' and 3' terminus of the RNA, a replicase binding site, and the structure of the coat protein cistron start. Some of these features resemble MS2 type coliphages but others the Q beta type. These findings suggest that PP7 is related to the coliphages but branched off before the coliphages diverged into separate groups.

Amino Acid Sequence↗
PubMed2004

Synthesis of dimeric trifluoromethoxyacridine-derived pathogen-inactivating nucleic acid intercalators.

A series of antiviral active compounds consisting of an intercalating acridine-derived part, a spacer region and a reactive EDTA-derived conjugate was synthesized in an easy sequence. Thus, suitably mono-protected 1,omega-alkyldiamines gave, upon reaction with 9-chloro-2-trifluoromethoxyacridine, followed by deprotection and reaction with EDTA dianhydride, the target molecules. Incorporation of their Fe(II) complexes in the presence of ascorbate gave a reduction of the phage titer of MS2 phages by several logarithmic decades.

Acridines↗
PubMed2007

Solubilization and stabilization of bacteriophage MS2 in organic solvents.

Several techniques were examined for the solubilization of bacteriophage MS2 in organic solvents. Direct extraction of the MS2 from an aqueous phase into isooctane containing 2 mM AOT, a proven approach for the organic solubilization of many proteins, was not successful. However, predried samples of MS2 were solubilized through the direct addition of organic solvents containing 500 mM AOT. As an alternative procedure, reverse micelles containing aqueous solutions of MS2 were prepared in isooctane using AOT, dehydrated through solvent evaporation and azeotropic drying, and resolubilized in a solvent of choice. The structure and microenvironment of organic-solubilized MS2 were investigated by UV absorbance, the fluorescence emission of an attached solvatochromatic dye, tryptophan fluorescence, and atomic force microscopy, all of which contributed evidence for a fully assembled capsid in the organic solvent. The solubilized MS2 was derivatized with stearic acid in chloroform, illustrating that bioconjugation reactions can be performed on organic-solubilized capsids using reagents that are completely insoluble in water. Furthermore, the organic-solubilized phage remained infectious after heating at 90 degrees C for 20 min, whereas phage in aqueous buffer or dried with nitrogen were nonviable following the heat treatment protocol. The extended range of available chemical modifications and the enhanced thermal stability of the organic-solubilized capsids bodes well for the formulation of storage-stable vaccines predicated on reactions in or exposure to organic media.

Biotechnology↗
PubMed2005

Behaviors of the MS2 virus and related antibodies in capillary isoelectric focusing with whole-column imaging detection.

Capillary isoelectric focusing (CIEF) has potential importance for the study of viruses. CIEF with whole-column imaging detection (WCID) is a novel CIEF mode, providing the advantages of high resolution, high speed, and easy method development. To facilitate the application of CIEF-WCID to the immunoassay of viruses, a basic knowledge of related aspects is necessary. In this study, the MS2 bacteriophage was used as a virus model, and the behaviors of MS2 and related antibodies in CIEF were investigated with UV absorbance-WCID and laser-induced fluorescence (LIF)-WCID. The adsorption of the virus and antibodies on the capillary wall was found to be the critical issue in method development. Addition of salt was found to be an effective way to reduce the adsorption and to improve peak shape. The formation of an immunocomplex, which forms the basis of an immunoassay, was monitored with CIEF-WCID. In comparison with UV-WCID, LIF-WCID was advantageous due to its higher detection sensitivity and the elimination of precipitation. Utilization of the noncovalent fluorescent dye, NanoOrange, was demonstrated to be a potential approach for the fluorescent labeling of the virus model and antibody and the associated immunocomplex. The change in microheterogeneity during the immune interactions at different ratios was also observed.

Adsorption↗
PubMed1994

Leeway and constraints in the forced evolution of a regulatory RNA helix.

The start of the coat protein gene of RNA phage MS2 adopts a well-defined hairpin structure of 12 bp (including one mismatch) in which the start codon occupies the loop position. An earlier expression study using partial MS2 cDNA clones had indicated that the stability of this hairpin is important for gene expression. For every -1.4 kcal/mol increase in stability a 10-fold reduction in coat protein was obtained. Destabilizations beyond the wild-type value did not affect expression. These results suggested that the hairpin was tuned in the sense that it has the highest stability still compatible with maximal ribosome loading. Employing an infectious MS2 cDNA clone, we have now tested the prediction that the delta G 0 of the coat protein initiator helix is set at a precise value. We have introduced stabilizing and destabilizing mutations into this hairpin in the intact phage and monitored their evolution to viable species. By compensatory mutations, both types of mutants quickly revert along various pathways to wild-type stability, but not to wild-type sequence. As a rule the second-site mutations do not change the encoded amino acids or the Shine-Dalgarno sequence. The return of too strong hairpins to wild-type stability can be understood from the need to produce adequate supplies of coat protein. The return of unstable hairpins to wild-type stability is not self-evident and is presently not understood. The revertants provide an evolutionary landscape of slightly suboptimal phages, that were stable at least for the duration of the experiment (approximately 20 infection cycles).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1995

Crystallization of the MS2 translational repressor alone and complexed to bromouridine.

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.

Bromodeoxyuridine↗
PubMed2002

Rapid microorganism identification with on-slide proteolytic digestion followed by matrix-assisted laser desorption/ionization tandem mass spectrometry and database searching.

A simple method for microorganism identification is proposed in this paper. In this method, no isolation or fractionation of microorganisms and no special search algorithm or database are needed. Experimentally, a sample of the unfractionated intact microorganism is subjected to brief on-slide proteolytic digestion and the digestion product ions detected in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) are selected for tandem mass spectrometry (MS/MS). Database searching using the MS/MS spectrum identifies the microorganism protein, and thereby, according to the source of the protein, the microorganism. The method was demonstrated to be feasible with enterobacteriophage MS2 as the model microorganism. The novel method is simple, rapid, and requires a small quantity of sample.

Amino Acid Sequence↗
PubMed1997

Subunit fusion confers tolerance to peptide insertions in a virus coat protein.

An octapeptide sequence called Flag was inserted into the bacteriophage MS2 coat protein at two different locations and its effects on protein folding and virus assembly were determined. Assays of the translational repressor and capsid assembly functions of the recombinants show that when the peptide is inserted at its N-terminus coat protein folds properly into the form that binds RNA (i.e., the dimer), but is defective for capsid assembly. On the other hand, a recombinant protein which is expected to display the Flag insertion as a surface loop does not fold correctly and, as a consequence, is proteolytically degraded. Genetic fusion of the two subunits of the coat dimer results in a protein considerably more tolerant of these structural perturbations and mostly corrects the defects accompanying Flag peptide insertion. Increased resistance of the single-chain coat protein to urea denaturation indicates that the fused dimer is substantially more stable than wild type. Covalent joining of subunits of oligomers probably represents a general strategy for engineering increased protein stability.

Amino Acid Sequence↗
PubMed2000

Suppression of the humoral immune response of Atlantic salmon, Salmo salar L. by the 64 kDa serine protease of Aeromonas salmonicida.

Bacteria-free supernatants of broth cultures of Aeromonas salmonicida inhibited the humoral immune response, but not the cell-mediated immune response, of Atlantic salmon to bacteriophage MS2. The immunosuppressive factor was the 64 kDa serine protease secreted by A. salmonicida. The suppressive activity was not due to degradation of epitopes of MS2, and although serine protease degraded the heavy chain of salmon IgM in vitro there was no evidence for significant degradation in vivo. The principal lethal toxin of A. salmonicida, the glycerophospholipid: cholesterol acyltransferase did not inhibit the immune response of salmon.

Aeromonas↗
PubMed1993

The refined structure of bacteriophage MS2 at 2.8 A resolution.

Bacteriophage MS2 is an icosahedral virus with 180 copies of a coat protein forming a shell around a single-stranded RNA molecule. The coat protein subunits form a lattice with the triangulation number T = 3. The coat protein has a fold which is different from the fold of all other viral coat proteins so far known. It consists of a five-stranded beta sheet facing the inside of the particle, and a hairpin and two helices on the outside. The crystal structure has been refined at 2.8 A resolution. The final R-factor was 0.189 for reflections with F > 2 sigma, and the root-mean-square deviation from idealized bond lengths and bond angles was 0.015 A and 2.9 degrees, respectively. The three chemically identical conformers A, B and C are largely similar. The B conformer has a unique conformation in one loop, which is involved in 5-fold interactions, while the A and C conformers, which are involved in the quasi-6-fold contacts, are similar throughout the structure. One cis-proline has been identified in the B conformer but the corresponding prolines in A and C are of the trans isomer. This residue is conserved within small RNA coliphages and it is proposed that this isomerization enables a less elongated loop (FG) around the 5-fold axis, thus creating a channel. The extensive dimer contact supports the idea of dimers as initial building blocks. An assembly pathway is proposed where five dimers converge into a pentamer and 12 pentamers are linked together with free dimers creating a complete particle.

Amino Acid Sequence↗
PubMed1994

Crystal structure of bacteriophage fr capsids at 3.5 A resolution.

The structure of recombinant capsids of the bacterial virus fr has been determined by X-ray crystallography at 3.5 A resolution. The capsids were produced by expressing the fr coat protein in Escherichia coli, the natural host of the virus, and are probably essentially identical to the protein shell of the native virus. The structure was determined using molecular replacement with the protein shell of the related MS2 virus, and refined to a crystallographic R-factor of 0.228. A comparison of the protein shells of the viruses shows that they are very similar, and indicates that they may have a similar regulation of the assembly of the quasi-symmetrical protein shell.

Amino Acid Sequence↗