Requirement of Protein Synthesis for Bacteriophage φX174 Superinfection Exclusion

Within 5 to 10 min at 37 C, bacteria infected by bacteriophage phiX174 acquire the ability to exclude superinfecting phiX particles from reproducing themselves. The superinfecting phage are blocked at a stage prior to synthesis of the parental replicative form molecule; the superinfecting deoxyribonucleic acid remains as intact (infective) single strands. Establishment of superinfection exclusion and its maintenance require protein synthesis.     

Greater Vulnerability of the Infecting Viral Strand of Replicative-Form Deoxyribonucleic Acid of Bacteriophage φX174

Four types of phiX-infected cells of Escherichia coli CR, a thymine-requiring strain of E. coli C, were prepared in which the parental replicative-form deoxyribonucleic acid (RF DNA) was labeled with same specific amounts of bromouracil in (i) both strands, (ii) only the infecting viral strand, (iii) only the complementary strand, and (iv) neither strand. The sensitivity of each type of infected cell toward irradiation by ultraviolet light, visible light, and X rays was measured. The results indicate that a certain amount of radiation damage in the infecting viral strand of the parental RF was more inhibitory to the production of progeny phage than when the damage was in the complementary strand. Similar conclusions were also drawn from "suicide" experiments of the phage-infected complexes containing (32)P of the same specific activity on either strand of the parental RF DNA. The results suggest that the beta decay occurring in the infecting viral strand was more effective in inactivating the plaque-forming ability of the complex.     

Genetic Recombination in Bacteriophage φX174

Genetic recombination in bacteriophage X174 usually takes place early in the infection process and involves two parental replicative form (double-stranded) DNA molecules. The host recA protein is required; none of the nine known X174 cistron products is essential. The products of a single recombination event are nonreciprocal and asymmetric. Typically, only one of the parental genotypes and one recombinant genotype are recovered from a single cell. An alternative, less efficient recombination mechanism which requires an active X174 cistron A protein is observed in the absence of the host recA gene product.     

Mechanism of Replication of Single-Stranded φX174 DNA VII. Circularization of the Progeny Viral Strand

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.     

The nucleotide sequence of bacteriophage φX174

The complete nucleotide sequence of the DNA of bacteriophage φX174 has been determined. The provisional sequence (Sanger et al., 1977a) deduced largely by the plus and minus method, has been completed and confirmed, predominantly using the terminator method (Sanger et al., 1977b). About 30 revisions were found to be necessary in the 5386-nucleotide sequence. The amino acid sequences of the ten proteins for which the DNA codes have also been deduced.     

Mutant of φX174 Accessible to Host-controlled Modification

A proflavine-induced X174 mutant was isolated, X174 sB1, which is accessible to restriction and modification by B-host specificity, but not by K-host specificity.     

Mode of Host Cell Penetration by Bacteriophage φX174

Bacteriophage phiX174 is an icosahedral phage which attaches to host cells without the aid of a complex tail assembly. When phiX174 was mixed with cell walls isolated from the bacterial host, the virions attached to the wall fragments and the phage deoxyribonucleic acid (DNA) was released. Attachment was prevented if the cell walls were treated with chloroform. Release of phage DNA, but not viral attachment, was prevented if the cell walls were incubated with lysozyme or if the virions were inactivated with formaldehyde. Treatment of the cell walls with lysozyme released structures which were of uniform size (6.5 by 25 nm). These structures attached phiX174 at the tip of one of its 12 vertices, but the viral DNA was not released. The virions attached to these structures were oriented with their fivefold axis of symmetry normal to the long axis of the structure. No virions were attached to these structures by more than one vertex. Freeze-etch preparations of phiX174 adsorbed to intact bacteria showed that the virions were submerged to one half their diameter into the host cell wall, and the fivefold axis of symmetry was normal to the cell surface. A second cell could not be attached to the outwardly facing vertex of the adsorbed phage and thus the phage could not cross-link two cells. When the virions were labeled with (3)H-leucine, purified, and adsorbed to Escherichia coli cells, about 15% of the radioactivity was recovered as low-molecular-weight material from spheroplasts formed by lysozyme-ethylenediaminetetraacetic acid. Other experiments revealed that about 7% of the total parental virus protein label could be recovered in newly formed progeny virus.     

Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.     

Effect of Oxidized Polyamines on the Infectivity of φX174 DNA

Oxidized spermine and oxidized spermidine inhibited markedly the infectivity of the 6 m-urea treated φX174 particle, whereas they did not inactivate the infectivity of the untreated phage particle. They also markedly inhibited the infectivity of φX174 DNA, while φX174 RF I DNA was less sensitive to these reagents. These facts suggested that oxidized polyamines could react with phage DNA.

The possible reasons of the insensitivity of phage φX174 particle and less sensitivity of φX174 RF I DNA to these reagents were discussed.     

Photodynamic Inactivation of Antigenic Determinants of Single-Stranded DNA Bacteriophage φX174

Bacteriophage phiX174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with UV-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called "jacket" component of the phiX174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the "spikes" are either poor antigens or insensitive to photodynamic treatment.     

Process of Infection with Bacteriophage φX174: XXXIII. Templates for the Synthesis of Single-Stranded Deoxyribonucleic Acid

The origin of the templates for the synthesis of X174 progeny single-stranded deoxyribonucleic acid was studied by means of the mutagenic activity associated with the decay of incorporated ³H-labeled 5-cytosine. The results indicate that the single-strand synthesis occurs in an asymmetric semiconservative manner using as template the complementary strands of the pool of replicative from molecules accumulated during the eclipse period. These complementary strands are repeatedly used as templates, and there is no detectable preferential use of complementary strand templates made early in the eclipse versus those made late.     

Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The double-stranded replicative form deoxyribonucleic acid (RF-DNA) of bacteriophage phiX174 was fragmented by pancreatic deoxyribonuclease, and the complementary strand fragments were then annealed to intact viral single strands. When such complexes infected Escherichia coli spheroplasts, some of the progeny virus bore genetic markers derived from the RF-DNA fragments. In this way, genetic markers have been salvaged from DNA fragments less than 50 nucleotides in length. This method is potentially useful as a specific assay to aid in the purification of genetically defined DNA fragments and also as a mechanism for the incorporation of small chemically synthesized DNA sequences into viral genomes.     

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage φX174

Inactivation of φX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a φXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN₃ on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N₂ atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.     

Nature of φX174 Linear DNA from a DNA Ligase-Defective Host

Linear DNAs have been prepared from phiX phage and from phiX RF II (double-stranded circular form of phiX DNA, formed during infection and nicked in one or both strands) molecules derived from infection at the restrictive temperature of Escherichia coli ts7, a host mutant with a temperature-sensitive DNA ligase activity. The linear DNA from these phages can be circularized by annealing with fragments of phiX RF DNA produced by the Haemophilus influenzae restriction nuclease. The circularization experiment indicated that the site of breakage of the linear phage DNAs is not unique nor confined to a particular region of the genome. These linear DNAs were less than 0.1% as infective as circular phage DNA. The linear, positive strand of late RF II DNA, however, is uniquely nicked in the region of the phiX genome corresponding to cistron A. Although a low level of infectivity is associated with the linear DNA derived from late RF II, this infectivity appears to be a result of the association of linear positive and linear negative strands during the infectivity assay.     

Diffusion Constant and Dimension of Bacteriophage φX174 as Determined by Self-Beat Laser Light Spectroscopy and Electron Microscopy

The diffusion constant of bacteriophage phiX174 was determined by laser light self-beat spectroscopy. The method allows one to establish the diffusion constant in comparatively short time and with high accuracy. From the diffusion constant D(37) = 1.96 +/- 0.08 x 10(-7) cm(2)/s, the size of the virus particles within their aqueous milieu can be calculated. For phiX174, a diffusional diameter of 31.4 +/- 1.0 nm was found, in good agreement with measurements of diameters of freezeetched (32.3 +/- 1.8 nm) and negatively stained particles (33.8 +/- 2.1 nm), provided that the entire spikes of the virion are included. Other isometric viruses may show a complex interaction of the virion with the surrounding water and its ions.     

Mutations in cell division proteins FtsZ and FtsA inhibit φX174 protein-E-mediated lysis of Escherichia coli

Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites. These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites. Therefore, E. coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis. The ftsZ84 and the ftsA12 cell division mutant strains of E. coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division. The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division – rather than specific functions of cell division – plays an essential role in protein-E-mediated lysis. SulA-overproducing cells had a lysis-positive phenotype, the ring structure – but not the GTPase function - of FtsZ was impaired. Received: 14 April 1998 / Accepted: 9 June 1998     

Second-Site Suppressors of a Cold-Sensitive Prohead Accessory Protein of Bacteriophage φX174

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.