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cell wall lysin as a component of the bacteriophage phi 6 virion.cell wall lytic activity was found in particles of the lipid-containing bacteriophage ø6. the activity can be extracted from the virion with triton x-100 in the presence of salt. this treatment removes the membrane-like envelope of the virion which includes five proteins. the lysin requires detergent for in vitro activity. virus particles formed in nonsuppressor cells by several classes of ø6 nonsense mutants contained the lysin activity; however, particles formed by a mutant (unable to make pro ...1979469991
ultrastructure of bacteriophage phi 6: arrangement of the double-stranded rna and envelope.pseudomonas phaseolicola cells synchronously infected with phi 6 were fixed and embedded by several procedures. the diameter of phi 6 measured 75 nm and its nucleocapsid 60 nm. virus nucleocapsids were icosahedral and surrounded by a double membrane. in planar sections the nucleic acid appeared as a hexagonal ring and in cross section as two angular structures.1977874459
fitness of rna virus decreased by muller's ratchet.why sex exists remains an unsolved problem in biology. if mutations are on the average deleterious, a high mutation rate can account for the evolution of sex. one form of this mutational hypothesis is muller's ratchet. if the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations. in asexual populations, as muller noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the succe ...19902247152
tentative identification of rna-dependent rna polymerases of dsrna viruses and their relationship to positive strand rna viral polymerases.amino acid sequence stretches similar to the four most conserved segments of positive strand rna viral rna-dependent rna polymerases have been identified in proteins of four dsrna viruses belonging to three families, i.e. p2 protein of bacteriophage phi 6 (cystoviridae), rna 2 product of infectious bursa disease virus (birnaviridae), lambda 3 protein of reovirus, and vp1 of bluetongue virus (reoviridae). high statistical significance of the observed similarity was demonstrated, allowing identifi ...19892759231
function of pili in bacteriophage phi 6 penetration.the genome of bacteriophage phi 6, which has a lipid protein envelope, consists of three pieces of dsrna. virus infection is initiated by attachment to a phi 6-specific host pilus followed by fusion of the phage membrane and the bacterial outer membrane. in this study we analysed several different phi 6 hosts as well as more than 200 independently isolated phi 6-resistant variants derived from pseudomonas syringae pv. phaseolicola. it is shown that phi 6-specific pili are coded by genes located ...19852865329
the nucleocapsid of the lipid-containing double-stranded rna bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.the nucleocapsid of the enveloped double-stranded rna bacteriophage phi 6 was isolated by extraction with the nonionic detergent triton x-114 and subjected to disruption analysis with chelating and protein-denaturing agents. the subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. the role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid pr ...19873599179
differential sensitivities of viruses in red cell suspensions to methylene blue photosensitization.previous studies explored the feasibility of using the photosensitizer methylene blue (mb) as a virucidal agent in red cell suspensions. under treatment conditions (5 microm [5 mumol/l] mb, 3.4 x 10(4) j/m2) that resulted in more than 6 log10 inactivation of vesicular stomatitis virus (vsv) or of the enveloped bacteriophage phi 6, red cell membrane alterations were observed. increased red cell ion permeability and the binding of plasma proteins to the red cell surface were the most sensitive ind ...19948023394
determination of residual 4'-aminomethyl-4,5',8-trimethylpsoralen and mutagenicity testing following psoralen plus uva treatment of platelet suspensions.psoralens and uva light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. in order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (amt), we have investigated the residual levels and mutagenic potential of amt after uva phototreatment. 4'-aminomethyl-4,5',8-trimethylpsoralen, at a final concentration of 40 micrograms/ml, ...19938337254
preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue.all published reports have described methods for virus photoinactivation which significantly alter red cell (rbc) properties during storage. in order to improve virucidal activity and reduce damage to rbcs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. one compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization.19989709780
hybrid frequencies confirm limit to coinfection in the rna bacteriophage phi6.coinfection of the same host cell by multiple viruses may lead to increased competition for limited cellular resources, thus reducing the fitness of an individual virus. selection should favor viruses that can limit or prevent coinfection, and it is not surprising that many viruses have evolved mechanisms to do so. here we explore whether coinfection is limited in the rna bacteriophage phi6 that infects pseudomonas phaseolicola. we estimated the limit to coinfection in phi6 by comparing the freq ...19999971826
recombinant viral rdrps can initiate rna synthesis from circular templates.the crystal structure of the recombinant hepatitis c virus (hcv) rna-dependent rna polymerase (rdrp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. we made circularized rna templates and found that they were efficiently used by the hcv rdrp to synthesize product rnas that are significantly longer than the template, suggesting that rdrp ...200616373481
high-throughput analysis of growth differences among phage strains.although methods such as spectrophotometry are useful for identifying growth differences among bacterial strains, it is currently difficult to similarly determine whether bacteriophage strains differ in growth using high throughput methods. here we use automated spectrophotometry to develop an in vitro method for indirectly distinguishing fitness (growth) differences among virus strains, based on direct measures of their infected bacterial hosts. we used computer simulations of a mathematical mo ...201222101310
epistasis and its relationship to canalization in the rna virus phi 6.although deleterious mutations are believed to play a critical role in evolution, assessing their realized effect has been difficult. a key parameter governing the effect of deleterious mutations is the nature of epistasis, the interaction between the mutations. rna viruses should provide one of the best systems for investigating the nature of epistasis because the high mutation rate allows a thorough investigation of mutational effects and interactions. nonetheless, previous investigations of r ...200415238511
rna structure and heterologous recombination in the double-stranded rna bacteriophage phi 6.bacteriophage phi 6 has a genome of three segments of double-stranded rna, designated l, m, and s. a 1.2-kbp kanamycin resistance gene was inserted into segment m but was shown to be genetically unstable because of a high recombination rate between segment m and the 3' ends of segments s and l. the high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. removal of one arm of this potential hairpin stabilizes the insertion. the insertion of a 241- or 427-bp la ...19938331732
in vitro packaging of the single-stranded rna genomic precursors of the segmented double-stranded rna bacteriophage phi 6: the three segments modulate each other's packaging efficiency.bacteriophage phi 6 is a double-stranded rna (dsrna) virus that has a genome composed of three linear dsrna segments (l, m, s). these are encapsidated into a dodecahedral procapsid particle consisting of proteins p1, p2, p4 and p7. expression of the cdna copy of the l segment in escherichia coli leads to the formation of empty procapsid particles. these particles are able to package the plus-sense single-stranded rna (ssrna)s of each genome segment in vitro. we have used this in vitro system for ...19957877165
rna binding, packaging and polymerase activities of the different incomplete polymerase complex particles of dsrna bacteriophage phi 6.phi 6 is an enveloped dsrna bacterial virus. its segmented genome resides inside the virion associated polymerase complex which is formed by four proteins (p1, p2, p4 and p7) encoded by the viral l segment. complete and incomplete polymerase complex particles can be produced using cdna copies of this largest genome segment. we have analysed the capacity of the different purified particles to (1) package phi 6 (+) sense genomic precursors and unspecific rna, (2) synthesize (-) and (+) strands and ...19957783210
differentiation between minus- and plus-strand synthesis: polymerase activity of dsrna bacteriophage phi 6 in an in vitro packaging and replication system.empty procapsids of the segmented dsrna virus phi 6, produced in escherichia coli from a cloned l genome segment, package plus-strand phi 6 ssrna genomic segments, synthesize minus strands, and transcribe the newly formed dsrna templates. procapsids can be restricted to minus-strand synthesis by high concentrations of cacl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) rna-dependent rna polymerase activities in ...19957645229
[comparative study of the antiviral activity of natural double-stranded rnas in experimental tick-borne encephalitis].a comparative study of the antiviral effect of interferon inducers from the group of natural double-stranded rnas (yeast plasmid dsrna, phage phi 6 and phage f2 dsrnas was carried out on the model of experimental tick-borne encephalitis. the possibility of inducing up to 60% protection against 10 ld50 of tbe virus by prophylactic inoculation of interferon inducers alone was demonstrated. the therapeutic effect was observed only with yeast dsrna (30% protection when the inducer was administered 4 ...19854095977
membrane fusion in prokaryotes: bacteriophage phi 6 membrane fuses with the pseudomonas syringae outer membrane.protein-triggered membrane fusion in the prokaryotic system is described using the lipid-containing enveloped bacterial virus phi 6 and its host, the gram-negative bacterium pseudomonas syringae. bacteriophage particles can be fused to form multiple particles where two or more nucleocapsids are surrounded by a single membrane vesicle with a volume proportional to the number of fused particles. for fusion to occur, a fusogenic protein is required in the membrane of the participating phage particl ...19873608985
the nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane.bacteriophage phi 6 infects its host, the gram-negative bacterium pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane. in this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle. this indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously ...19883169005
chemical crosslinking of bacteriophage phi 6 nucleocapsid proteins.phi 6 is a lipid-containing dsrna bacteriophage of pseudomonas syringae. its nucleocapsid (nc) has common features with reoviridae core particles. we report here the crosslinking of phi 6 nc proteins with cleavable 12-a span chemical crosslinker, dithiobis(succinimidyl propionate). the crosslinked complexes were analyzed in two-dimensional polyacrylamide gels or by using monoclonal antibodies to uncleaved protein complexes in one-dimensional protein gels. the nc surface protein (p8) forms a seri ...19883043894
quantitation of the adsorption and penetration stages of bacteriophage phi 6 infection.the enveloped dsrna bacteriophage phi 6 uses the pilus of pseudomonas syringae as its receptor. it enters the host cell by fusion of the virus envelope with the host outer membrane, followed by penetration of the cytoplasmic membrane by the phage nucleocapsid. in this investigation we quantitated the adsorption and penetration of phi 6wt and a host range mutant, phi 6h 1s, to five bacterial strains. adsorption rate constants were measured for the different phage-host combinations, the constant f ...19892741342
[comparative study of interferon inducers obtained from natural sources].interferon-inducing and antiviral effects of natural dsrna preparations of phage phi 6 and yeast cells were studied in the culture of murine cells l-929 and on random bred albino mice. both the preparations showed interferon inducing activity in the cell culture. however, for realization of their effect modification of the surface cell membrane by polycation exchange resin (deae-dextran) was required. the interferon-inducing activity of both of the natural dsrna in the mice was high. the maximum ...19872436574
purified phi 6 nucleocapsids are capable of productive infection of host cells with partially disrupted outer membranes.purified nucleocapsids of bacteriophage phi 6, lacking the phage lipid envelope, are unable to infect intact pseudomonas syringae host cells. a method for studying the process by which a naked virus particle, the phi 6 nucleocapsid, penetrates the host cytoplasmic membrane was developed. host cells were rendered competent for nucleocapsid infection by treatment with repeated washings with salt and sucrose and the subsequent addition of lysozyme. this treatment disrupts the outer membrane, permit ...19902219699
construction of a transducing virus from double-stranded rna bacteriophage phi6: establishment of carrier states in host cells.bacteriophage phi 6 contains three double-stranded rna (dsrna) genomic segments. we have constructed a plasmid that contains a cdna copy of the middle (m) segment, with a gene for kanamycin resistance (kan) inserted into the psti site. a transcript of this cdna was incorporated in vitro into procapsids along with natural transcripts of the s and l segments. the procapsids were coated with nucleocapsid surface protein p8 and transfected into pseudomonas syringae pv. phaseolicola. the resulting in ...19921727482
bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy.bacteriophage phi 6 is an enveloped dsrna virus which infects the plant pathogenic pseudomonas syringae bacterium. using low dose cryoelectron microscopy we show that the nucleocapsid, spikeless virion, and intact virion have radii of 29, 35, and 43 nm, respectively. thus, the membrane is 6 nm thick and the surface spikes of the receptor binding protein p3 extend 8 nm from the membrane surface. cross-linking, immunological, and complementation evidence suggest that the spikes are formed of multi ...19921519356
the buoyant density of three double-stranded rnas in cesium sulfate.the buoyant densities in cs2so4 of the double-stranded rna from the bacteriophage phi 6 and the virus-like particles of helminthosporium maydis and penicillium chrysogenum were determined by a taylor series expansion and by the position and slope of the gradient relative to the isopycnic position (hinge point method). buoyant densities for the three types of double-stranded rna calculated by the two methods were, respectively, 1.6089 and 1.6083, 1.6065 and 1.6059 and 1.6057 and 1.6050.1976953002
ultrastructure and life cycle of the lipid-containing bacteriophage phi 6.an electron microscopic study of the lipid-containing bacteriophage phi 6 revealed an electron dense compact inner core of 30 nm in diam., which apparently contains the nucleic acid of the virus. this inner particle is surrounded by a complex polyhedral capsid with an outer diam. of 50 nm. outside this is the envelope, which gives the virus a total diam. of 65 to 75 nm. the envelope, which has a thickness of a unit membrane, could be removed by treating the phage with triton x-100. a definite st ...1976798023
assembly of the enveloped bacteriophage phi 6 in environments which perturb the host cell membranes.the effects of known membrane-perturbing agents (ph, na+, ca2+, and a small lipid-soluble molecule) on the enveloped bacteriophage phi 6 host cell system were investigated at the levels of cellular growth, virus assembly and stability, and the physical and chemical properties of host cell membranes. spin-label probes of cellular membranes indicate that growth in high levels of na+ or the small spherical hydrophobic molecule adamantanone results in membranes having increased "fluidity," while gro ...1975240501
pleiotropic costs of niche expansion in the rna bacteriophage phi 6.natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. when a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. here we investigate the evolution of expanded host range (generalism) in the rna virus phi6, an experimental model system allowing adaptive mutations to be readily identified. we ...200616299384
evolution of mutational robustness in an rna virus.mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. robustness should be selectively favored when mutation rates are high, a common feature of rna viruses. however, selection for robustne ...200516248678
rescue of maturation off-pathway products in the assembly of pseudomonas phage φ 6.many complex viruses use an assembly pathway in which their genome is packaged into an empty procapsid which subsequently matures into its final expanded form. we utilized pseudomonas phage 6, a well-established virus assembly model, to probe the plasticity of the procapsid maturation pathway. the 6 packaging nucleoside triphosphatase (ntpase), which powers sequential translocation of the three viral genomic single-stranded rna molecules to the procapsid during capsid maturation, is part of the ...201324089550
bacteriophage phi 6 rna-dependent rna polymerase: molecular details of initiating nucleic acid synthesis without primer.like most rna polymerases, the polymerase of double-strand rna bacteriophage phi6 (phi6pol) is capable of primer-independent initiation. based on the recently solved phi6pol initiation complex structure, a four-amino acid-long loop (amino acids 630-633) has been suggested to stabilize the first two incoming ntps through stacking interactions with tyrosine, tyr(630). a similar loop is also present in the hepatitis c virus polymerase, another enzyme capable of de novo initiation. here, we use a se ...200211877396
structural studies of the enveloped dsrna bacteriophage phi 6 of pseudomonas syringae by raman spectroscopy. i. the virion and its membrane envelope.we report and interpret the first raman spectrum of a double-stranded rna virus containing a membrane envelope. spectra of the native bacteriophage phi 6 and of its isolated host-attachment (spike) protein and phospholipid-free core assembly were collected from aqueous solutions over a wide range of temperature. comparison of the vibrational spectra by digital difference methods permits the following structural conclusions regarding molecular constituents of the fully assembled virion. (1) the d ...19938464060
protein p4 of double-stranded rna bacteriophage phi 6 is accessible on the nucleocapsid surface: epitope mapping and orientation of the protein.protein p4, an early protein of double-stranded rna bacteriophage phi 6, is a component of the virion-associated rna polymerase complex and possesses a nucleoside triphosphate (ntp) phosphohydrolase activity. we have produced and characterized a panel of 20 p4-specific monoclonal antibodies. epitope mapping using truncated molecules of recombinant p4 revealed seven linear epitopes. the accessibility of the epitopes on the phi 6 nucleocapsid (nc) surface showed that at least the c terminus and an ...19937682630
in vitro transcription of the double-stranded rna bacteriophage phi 6 is influenced by purine ntps and calcium.the double-stranded rna bacteriophage phi 6 contains a virion-associated rna-dependent rna polymerase complex. removal of the virus envelope and the nucleocapsid surface protein, p8, reveals a nucleocapsid core particle (proteins p1, p2, p4, p7) which is the viral polymerase complex, capable of synthesizing rna strands of positive polarity. the in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation ...19957886944
the polymerase subunit of a dsrna virus plays a central role in the regulation of viral rna metabolism.bacteriophage φ6 has a three-segmented double-stranded (ds) rna genome, which resides inside a polymerase complex particle throughout the entire life cycle of the virus. the polymerase subunit p2, a minor constituent of the polymerase complex, has previously been reported to replicate both φ6-specific and heterologous single-stranded (ss) rnas, giving rise to dsrna products. in this study, we show that the enzyme is also able to use dsrna templates to perform semi-conservative rna transc ...200011080173
genomic and gene-expression comparisons among phage-resistant type-iv pilus mutants of pseudomonas syringae pathovar phaseolicola.pseudomonas syringae pv. phaseolicola (pph) is a significant bacterial pathogen of agricultural crops, and phage φ6 and other members of the dsrna virus family cystoviridae undergo lytic (virulent) infection of pph, using the type iv pilus as the initial site of cellular attachment. despite the popularity of pph/phage φ6 as a model system in evolutionary biology, pph resistance to phage φ6 remains poorly characterized. to investigate differences between phage φ6 resistant pph strains, we examine ...201526670219
single-molecule measurements of viral ssrna packaging.genome packaging of double-stranded rna (dsrna) phages has been widely studied using biochemical and molecular biology methods. we adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. to our knowledge, this is the first attempt to study the details of viral rna packaging using optical tweezers. pseudomonas phage φ6 is a dsrna virus with a tripartite genome. positive-sense (+) single-stranded rna (ssrna) genome precursors are packaged into a prefor ...201727803153
prisoner's dilemma in an rna virus.the evolution of competitive interactions among viruses was studied in the rna phage phi6 at high and low multiplicities of infection (that is, at high and low ratios of infecting phage to host cells). at high multiplicities, many phage infect and reproduce in the same host cell, whereas at low multiplicities the viruses reproduce mainly as clones. an unexpected result of this study was that phage grown at high rates of co-infection increased in fitness initially, but then evolved lowered fitnes ...199910201376
cumulative viral titer reduction demonstrated by sequential challenge of a tangential flow membrane filtration system and a direct flow pleated filter cartridge.the efficiency of sequential incorporation of two approaches to virus removal, i.e., tangential flow ultrafiltration using the omega 300k vr maximate system, and, direct flow microfiltration using the ultipor vf grade dv50 virus removal filter, was evaluated using bacteriophage phi 6 in dulbecco's modified eagle medium (mem) as the carrier fluid. a combined log titer reduction value of > 10 was demonstrated, with each step, individually, providing > 4.5 log reduction. significant improvement in ...20169357307
heterologous recombination in the double-stranded rna bacteriophage phi 6.bacteriophage phi 6 contains three double-stranded rna genomic segments. we have constructed a virus with an insertion of a kanamycin resistance gene in genomic rna segment m. the virus forms small, turbid plaques, and its genome is unstable. virus from a single plaque contained from about 0.1 to 10% large clear-plaque forms of the virus; these were usually missing the kanamycin resistance gene, and in many cases, the resulting segment m was larger or smaller than its normal size. sequence analy ...19921560519
nucleotide sequence of the large double-stranded rna segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase.the genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded rna. we determined the nucleotide sequence of cdna derived from the largest rna segment (l). this segment specifies the procapsid proteins necessary for transcription and replication of the phi 6 genome. the coding sequences of the four proteins on this segment were identified on the basis of size and the correlation of predicted n-terminal amino acid sequences with those found through analysis of i ...19883346944
bacteriophage phi 6: a unique virus having a lipid-containing membrane and a genome composed of three dsrna segments. 19883068964
mechanism of virucidal activity of retinoids: protein removal from bacteriophage phi 6 envelope.the mechanism of the potent virucidal activity of retinol (vitamin a) and retinal (vitamin a aldehyde) was investigated for the interaction of these compounds with a model system enveloped virus, bacteriophage phi 6. the retinoids bind to and inactivate phi 6 without causing large-scale disruption of the virion. inactivated virions have completely lost the ability to adsorb to host bacteria due to the loss of a viral envelope protein necessary for adsorption.19807406477
in vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded rna virus.a system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. procapsids encoded by cdna copies of genomic segment l in escherichia coli were used to package and replicate viral rna segments. the resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein p8. the infected spheroplasts yielded infectious virions. a modified cdna-derived rna segment was inserted into virions by this ...19902251260
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