Structure and synthesis of a lipid-containing bacteriophage. XIX. Reconstitution of bacteriophage PM2 in vitro.

In order to construct a physical map of the bacteriophage fd genome, the doubly closed replicative form (RFI) DNA of phage fd was cleaved into unique fragments by four different restriction endonucleases (Hap, Hga, HinH and Hind) prepared from Haemophilus strains H. aphirophilus, H. gallinarum, H. influenzae H-I and H. influenzae Rd, respectively. As Hind cleaved RFI DNA at a single site, this site was used as a reference point for mapping. HinH cleaved RFI DNA at three sites, Hga at six sites and Hap at 13 sites, respectively. The 5′-termini of the fragments produced by either HinH or Hga were labelled with ³²P in the polynucleotide kinase reaction. The labelled fragments were separated and further cleaved by other enzymes. The re-digestion products of partially digested fragments were also analysed. On the basis of these data and estimates of the size of each fragment, a cleavage map of the phage fd genome was constructed.     

Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.     

Structure and synthesis of a lipid-containing bacteriophage. Amphiphilic properties of protein IV of bacteriophage PM2

Interactions between lipids and the DNA-binding protein (protein IV) purified from bacteriophage PM2 were studied in vitro. The efficiency of incorporation of protein IV into single-walled liposomes was more than 90%. Protein IV embedded in liposomes interacted more strongly with PM2 DNA than protein IV alone. The DNA--protein-IV--liposome complex was relatively stable as observed by sedimentation behavior on a sucrose gradient. The interaction between DNA and the protein-IV--liposome was abolished by tryptic digestion, even though 40% of the protein remained in the vesicle. More than 70% of the amino acids of this embedded peptide segment were hydrophobic. Carboxypeptidase digestion of the protein-IV--liposome caused a release of 20% of the radioactivity of the vesicle without changing the DNA-binding ability of the complexes. Modification of the protein-IV--liposome with the chemical probe, 2,4-dinitrofluorobenzene, and analysis of the tryptic peptides released from the protein-IV--liposome demonstrated that the N-terminal basic amino acid cluster segment responsible for the DNA binding was located on the outer surface of the bilayer. These results support an earlier model in which protein IV anchors itself in the inner leaflet of the PM2 bilayer membrane, interacting with the DNA in the virion.     

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4.

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.     

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.     

Physical measurements on the lipid-containing bacteriophage φ6

Bacteriophage φ6 has been studied by small-angle X-ray scattering, intensity-fluctuation spectroscopy, analytical ultracentrifugation, and spectroscopy. The sedimentation coefficient (s₂₀⁰, w) is 375 S, the diffusion coefficient (D₂₀⁰, w) is 2.66 · 10^?8 cm²/s. Using the Svedberg equation and an estimate of the partial specific volume, the M_r is 1.49 ± 0.32 · 10⁸.A simple model which describes φ6, is a central sphere consisting of RNA and protein of radius 330 Å and an outer shell of low electron density 40 Å thick. The RNA may form five concentric shells in the region $\text{r = 140?290}\text{A}\text{?}$     

Origin of the phospholipids of a lipid-containing virus that replicates in Escherichia coli: bacteriophage PR4.

Bacteriophage PR4 contains lipid and can reproduce in strains of Escherichia coli that carry an appropriate drug-resistance plasmid. Cultivated in either of two E. coli strains, PR4 acquires a lipid region that contains a relatively high level of phosphatidylglycerol and significant amounts of three phospholipids, including phosphatidylserine, which are present in only very low levels in the host cell membranes. To do this, however, PR4 does not significantly alter the relative levels of synthesis of the various E. coli phospholipids after infection. Production of PR4 virions from E. coli cultures labeled with 32PO4 either before or after infection showed that about two-thirds of the viral phospholipid is synthesized after infection. The use of E. coli as the host organism for PR4 should allow a detailed understanding of the assembly process of this lipid-containing virus due to the wealth of biochemical and genetic techniques available.     

Phospholipid metabolism in Pseudomonas BAL-31 infected with lipid-containing bacteriophage PM2.

Infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2 resulted in no detectable change in the rate of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) biosynthesis. An increase in the PG content of infected cultures was not seen until the cultures began to lyse, and this increase was in fact only a relative increase resulting from the extensive turnover of PE at the onset of culture lysis. Turnover studies revealed that the glycerol, phosphorus fatty acid, and ethanolamine moieties of PE turned over simultaneously at the time of lysis, and therefore made it unlikely that there was a PE to PG conversion during the latent period of the phage. The lipid found in the bacteriophage did not reflect a preferential selection for lipid synthesized before or after infection, but in fact reflected the composition of the host membrane at the time the phage were assembled. The use of a modified medium that allowed the cultivation of Pseudomonas BAL-31 as a prototroph and resulted in reliable lysis times of infected cultures led us to the conclusion that PM2 infection effects little change in host phospholipid metabolism, and that there is sufficient PG in the host cytoplasmic membrane to account for a full burst of phage. As a result of the reliable lysis times that we have achieved, we concluded that certain metabolic events, i.e., PE turnover, are lytic phenomena and must not be confused with events relevant to the biosynthesis and maturation of the phage.     

Calcium requirement for assembly of the lipid-containing bacteriophage PM2

The bacteriophage PM2 requires extracellular Ca²⁺ at concentrations greater than 3 · 10⁻⁴ M for the production of viable virus, whereas the host cell Pseudomonas BAL-31 grows normally in medium containing 3 · 10⁻⁵ M Ca²⁺ (low calcium). Virus attachment occurs normally in low calcium, the infected cultures partially lyse, but no infectious virus particles are released. Sucrose gradient analysis shows that lysates made in low calcium contain no PM2-like particles. The addition of calcium very late in the infectious cycle completely restores virus production to cultures infected in low calcium, whereas removal of calcium after infection prevents virus production. Our experiments indicate that Ca²⁺ is essential for some process late in the lytic cycle, such as the final assembly of stable, infectious PM2 particles.     

Inhibition of entry of the lipid-containing bacteriophage PR4 by fatty acid derivatives.

Amphiphilic fatty acid derivatives having a 14- to 20-carbon cis-unsaturated alkyl chain were found to be potent inhibitors of the entry process of the lipid-containing bacteriophage PR4.     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.     

Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.     

Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.     

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.     

Purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1.

Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an alpha-helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self-quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6-M guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.