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 proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.     

Study of the circular dichroism of bacteriophage phi 6 and phi 6 nucleocapsid

We have studied the CD of both bacteriophage ?6 and ?6 nucleocapsid in order to assess the in situ state of the viral, double-stranded RNA (dsRNA). The results showed that packaged ?6 RNA is slightly hyperdichroic (4–5%) at 264 nm relative to dsRNA in solution. Also, the CD of dsRNA within both the virus and the nucleocapsid was unlike CD spectra of any of the three types of dsRNA condensed in vitro, as described in the accompanying paper. Using a curve-fitting program described elsewhere (Edmondson, S.P. & Gray, D.M. (1983) Nucleic Acids Res. 11 , 175–192), we have fit reference CD spectra of ?6 RNA and protein to measured CD spectra of ?6 intact phage and nucleocapsid. Results of the computer analysis indicated that ?6 RNA in situ is spectrally similar to dsRNA that is slightly dehydrated.     

Plasmid-directed assembly of the lipid-containing membrane of bacteriophage phi 6.

The nucleocapsid of bacteriophage phi 6 is enveloped within a lipid-containing membrane. The membrane is composed of proteins P3, P6, P9, P10, and P13 and phospholipids. The relationship between membrane protein P9 and morphogenetic protein P12 was studied in the absence of phage infection. cDNA copies of genes 9 and 12 were expressed on plasmids in Pseudomonas syringae pv. phaseolicola. Immunoblotting demonstrated the presence of protein P9 in strains carrying both gene 9 and gene 12 but not in strains with gene 9 alone. In the absence of P12, P9 was found to be unstable. Simultaneous synthesis of proteins P9 and P12 led to the formation of a low-density P9 particle having a buoyant density similar to that of precursor structures composed of phospholipid and proteins isolated from phi 6-infected cells. These results are consistent with results of previous genetic experiments suggesting that P9 and P12 are necessary and sufficient for the formation of the phi 6 envelope. Extensions of P9 at the C terminus do not impair particle formation; however, N-terminal extensions or C-terminal deletions that extend into the hydrophobic region of P9 do impair particle formation.     

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 expected. Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells. The penetration is dependent on the energized state of the host cytoplasmic membrane. About 25% of the entering core particles are re-used in the progeny viruses.     

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.     

Defective bacteriophage MS2 particles containing specific RNA FRAGMENTS]

Two types of MS2 particle are revealed when phage lysates are banded in CsCl density gradient. The lower band contain normal phage particles with a density of 1.46 g/cm3. The upper band with a density of 1.44 g/cm3 containes uninfective incomplete MS2 particles. Both phage types reveal no abnormalities in the content of the coat protein and A-protein. They are nearly identical in RNA content. RNA in the normal buoyant density phage particles is native. RNA in the defective particles consists of three specific fragments with molecular weights 6.5-10(5), 5.5-10(5) and 4.4-10(5) and molar ratios 5:4:9 respectively. THE 5'-TERMINAL ANALYSIS OF RNA from defective MS2 particles reveals the presence of native pppGp. THE 3'-TERMINAL ANALYSIS OF THE INDIVIDUAL RNA fragments reveals the presence of adenosine only in the shortest fragment. RNA fragmentation in defective particles can be explained by the action of intracellular RNAses on the unprotected regions on RNA chain in structurally incomplete virions.     

In vitro translation of the three bacteriophage phi 6 RNAs.

In vitro translation of the three single-stranded RNAs transcribed in vitro by bacteriophage phi 6 RNA polymerase revealed that the large RNA codes for phage proteins P1, P2, P4, and P7, the medium RNA codes for P3, P6, and P10, and the smaller RNA for P5, P8, and P9.     

Bacteriophage phi6: a Lipid-Containing Virus of Pseudomonas phaseolicola

The purification and properties of a lipid-containing bacteriophage, 6, are described. The phage contains a lipid envelope which is probably essential for infection. Infectivity of 6 was lost in the presence of organic solvents, sodium deoxycholate, and phospholipase A. The fatty acid composition of the phage lipid was similar to that of the Pseudomonas phaseolicola host cells. The phage was composed of about 25% lipid, 13% RNA, and 62% protein. The buoyant density of 6 was 1.27 g/ml in cesium chloride. The morphology of 6 was unusual; it had a polyhedral head of about 60 nm surrounded by a membranous, compressible envelope which appeared to assume an elongated configuration upon attachment to pili. The adsorption rate constant was 3.3 × 10⁻¹⁰ ml/min in a semi-synthetic medium and 3.8 × 10⁻¹⁰ ml/min in a nutrient broth-yeast extract medium. The latent period was shorter in the former medium (80-115 min compared with 120-160 min), and the average burst size was larger (250-400 compared with 125-150). The eclipse period coincided with the latent period.     

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.     

Characterization of segmented double-helical RNA from bacteriophage phi6

The nucleic acid component of bacteriophage φ6 is characterized as a double stranded RNA molecule with a buoyant density of 1.605 g/cm³ and nucleotide composition of C, 27.3%; A, 21.8%; G, 28.9%; and U, 22.0%. The hyperchromicity profile in 0.1 × SSC (SSC is 0.15 m-NaCl, 0.015 m-sodium citrate) demonstrated a rapid increase with a T_m value of 91 °C. The nucleic acid was resistant to degradation by DNase, spleen phosphodiesterase and pancreatic RNase in 2 × SSC buffer but sensitive to degradation by venom phosphodiesterase, pancreatic RNase in 0.01 × SSC and hydrolysis in KOH. Three distinct double stranded RNA species of 2.2, 2.8 and 4.5 × 10⁶ daltons were observed after rate zonal centrifugation, polyacrylamide gel electrophoresis and electron microscopy. This communication therefore presents data establishing a new class of double stranded RNA bacteriophage.     

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

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 internal domain, containing the consensus sequence for NTP binding, protrude the NC shell. Four of the NC-binding antibodies distorted the integrity of the NC by releasing protein P4 and the major NC surface protein P8. This finding suggests a close contact between these two proteins. The dissociation of the NC led to the activation of the virion-associated RNA polymerase. The multimeric status of the recombinant P4 was similar to that of the virion-associated P4, indicating that no accessory virus proteins are needed for its multimerization.     

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 isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous mutations in the phi6 gene P3, important in host attachment. Seven of these nine mutations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in phi6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses.     

Mechanism of Ozone Inactivation of Bacteriophage f2

The inactivation kinetics of bacteriophage f2 were studied by using ozone under controlled laboratory conditions. The phage were rapidly inactivated during the first 5 s of the reaction by 5 and 7 logs at ozone concentrations of 0.09 and 0.8 mg/liter, respectively. During the next 10 min, the phage were further inactivated at a slower rate in both treatments. The [³H]uridine-labeled f2 phage and its ribonucleic acid (RNA) were examined to elucidate the mechanism of ozone inactivation, utilizing adsorption to host bacteria, sucrose density gradient analysis, and electron microscopy. The specific adsorption of the phage was reduced by ozonation in the same pattern as plaque-forming unit reduction. RNA was released from the phage particles during ozonation, although it had reduced infectivity for spheroplasts. Electron microscopic examination showed that the phage coat was broken by ozonation into many protein subunit pieces and that the specific adsorption of the phage to host pili was inversely related to the extent of phage breakage. The RNA enclosed in the phage coat was inactivated less by ozonation than were whole phage, but inactivated more than naked RNA. These findings suggest that ozone breaks the protein capsid into subunits, liberating RNA and disrupting adsorption to the host pili, and that the RNA may be secondarily sheared by a reduction with and/or without the coat protein molecules, which have been modified by ozonation.     

Ribonucleic Acid Polymerase Activity in Sendai Virions and Nucleocapsid

After dissociation of purified Sendai virus with the neutral detergent Nonidet P-40 and 2-mercaptoethanol, it catalyzed the incorporation of ribonucleoside triphosphates into an acid-insoluble product. The enzyme activity was associated with viral nucleocapsid as well as whole virions. The reaction product was ribonucleic acid (RNA) which annealed specifically with virion RNA. Sedimentation of the (3)H-RNA reaction product revealed two components, a 45S component with properties of double-stranded RNA and 4 to 6S component which appeared to be mostly single-stranded RNA.     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.     

Properties of the Deoxyribonucleic Acid Contained in the Defective Particle Coliphage 15

Escherichia coli strain 15 TAU, which requires thymine, arginine, and uracil for growth and harbors an apparently defective prophage, was induced by exposure to ultraviolet light (580 ergs/mm(2)) or to mitomycin C (5 mug/ml). Phage particles (coliphage 15) were recovered from the resulting lysate by treatment with deoxyribonuclease, filtration, and several cycles of differential centrifugation. Analysis of the phage particles obtained by using cesium chloride density gradient centrifugation in a preparative ultracentrifuge resulted in the resolution of three components. The major component had a peak density of 1.52 to 1.53 g/cm(3) followed by components with densities of 1.5 and 1.49 g/cm(3). The guanine plus cytosine content of coliphage 15 deoxyribonucleic acid (DNA) was determined by both analytical ultracentrifugation in cesium chloride and by thermal denaturation in standard saline citrate buffer. Respective values of 46.4 +/- 1% and 46.6 +/- 1% guanine plus cytosine content were obtained. Coliphage 15 DNA formed molecular hybrids with messenger ribonucleic acid (RNA) from both uninduced and ultraviolet-induced cultures of E. coli 15 TAU, but did not hybridize with E. coli ribosomal RNA. The molecular weight of coliphage 15 DNA was determined by constant velocity sucrose density gradient centrifugation to be about 33 x 10(6) daltons.