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PubMed1976

Purification and properties of bacteriophage phi X 174 gene D product.

Bacteriophage phi X 174 gene D product, a protein required for single-stranded DNA synthesis by the phage, has been purified to near homogeneity. The protein is very abundant; approximately 10(5) monomers are present per infected cell when lysis is delayed. The protein has a monomer molecular weight of 15,200 and is normally a tetramer; however, it can form very large aggregates at high concentrations. Amino acid analysis shows an excess of arginine over lysine and a relatively high number of nonpolar residues. The protein carries a net negative charge at neutral pH. The first eight amino acids of the protein sequence have been determined; they are Ser-Gln-Val-Thr-Glu-Gln-Arg-Val. The carboxy-terminal residue is methionine. The protein has not yet been shown to bind directly to any single-stranded DNA; it does not adsorb to denatured calf thymus DNA-cellulose.

Amino Acid Sequence↗
PubMed1985

Antibody to bacteriophage phi X 174 synthesized by cultured human peripheral blood lymphocytes.

Following immunization of human subjects with the antigen bacteriophage phi X 174, concomitant with the rise in serum antibody, cells appear in the circulation which in vitro, without antigen stimulation, synthesize antibody of the same class as serum antibody in most subjects studied. This function is inhibited by puromycin or irradiation, is independent of T cells and occurs within the first 36-72 h of culture. Such cells are found only in recently immunized subjects. Peripheral blood mononuclear cells (PBM) from all immunized subjects synthesize more antibody to phi X 174 in vitro if antigen is present during cell culture; none was synthesized by antigen containing PBM cultures from unimmunized subjects. This antigen-induced antibody response is T cell and antigen dose-dependent and inhibited by puromycin or irradiation. Following primary immunization the antibody synthesized in vivo and in vitro is IgM. Following secondary immunization IgG antibody is immediately detected in vivo but in vitro antigen-induced antibody continues to be IgM for at least 3 months. IgG antibody then appears: once this class switch occurs, in vitro antigen-induced IgG antibody can be demonstrated in cultured PBM of subjects for many years, without further booster immunization.

Adult↗
PubMed1981

Control of bacteriophage phi X 174 gene expression by the transcription termination factor rho in vitro.

The effect of rho factor on bacteriophage phi X 174 gene expression was studied in a cell-free, DNA-dependent, RNA-directed protein synthesizing system (transcription-translation coupled system) using S30 extracts of wild-type and rho-mutant Escherichia coli cells. It has been found that in the presence of a functionally active and endogenous or added rho factor, only a limited number of the viral genes (A, B, and D) are expressed effectively, whereas the expression of genes, F, G, and H, which encode capsid proteins, is strongly inhibited. Mutational or thermal inactivation of rho factor results in a considerable activation of capsid protein synthesis and, at the same time, in some depression of the synthesis of gene A, B, and D products. From the results of this study it is suggested that phage phi X 174 has two classes of gene, late and early, which are separated on the chromosome by a rho-dependent transcription terminator. We propose that the expression of these classes is regulated by a termination-antitermination mechanism.

Bacteriophage phi X 174↗
PubMed1976

Process of infection with bacteriophage phi X 174 XXXVIII. Replication of phi chi 174 replicative form in vivo.

The replication of bacteriophage phi X 174 replicative-form DNA has been studied by structural analysis of pulse-labeled replicative-intermediate molecules. Such intermediates were identified by pulse-labeling with [13H]thymidine and separated into four major fractions (A, B, C, and D) in a propidium diiodide-cesium chloride buoyand density gradient. Sedimentation analysis of each of these fractions suggests the following features of phi X replicative-form DNA replication in vivo. (i) At the end of one cycle of replication, one daughter replicative form (RFII) contains a nascent plus (+) strand of the unit viral length, and the other daughter RFII contains small fragments of nascent minus (-) strand. (ii) Asymmetry is also associated with production of the first supercoiled RFI after addition of pulse label in that only the minus strand becomes radioactive. (iii) A supercoiled DNA (RFI') seems to occur in vivo. This DNA is observed at a position of greater density in a propidium diiodide-cesium chloride buoyant density gradient than normal RFI. (iv) A novel DNA component is observed, at a density greater than RFI, which releases, in alkali, a plus strand longer (1.5 to 1.7 times) than the unit viral length. These results are discussed in terms of the possible sequence of events in phi X 174 replicative-form replication in vivo.

Coliphages↗
PubMed1962

Antibody formation. III. The primary and secondary antibody response to bacteriophage phi X 174 in guinea pigs.

Injection of a small bacteriophage phiX 174 into guinea pigs results in an accelerated elimination of phage detectable as early as 24 hours after injection. The immune nature of the accelerated elimination is indicated by its specificity, by the appearance of excess specific serum antibody after phage elimination, and by the prevention of accelerated elimination by 400 r whole body x-irradiation of guinea pigs prior to injection of phage. The early antibody response is considered to be a primary one since an analogous response occurs in newborn guinea pigs, antibody is not detectable in the sera of non-immunized animals, and the second challenge with phiX stimulates a serum antibody response 100-fold greater than that after primary immunization. The early detection of immune elimination appears to be due, in part, to the small amounts of phage employed, since larger doses of phage delay the time of onset of detectable immune elimination. The early rise of serum antibody in the primary and secondary response appears exponential with a similar rate constant of antibody formation. The rate constant is also independent of dose. These findings have led to the suggestion that during this exponential phase, the relative rate of antibody formation at a cellular level may be constant for a given antigen.

Animals↗
PubMed1981

Disinfecting properties of performic acid against bacteriophage phi X 174 as a model of small envelope--free viruses.

Performic acid HCOOH (PFA) is a wide-spectrum disinfectant. It inactivates viruses, bacteria and bacterial spores, mycobacteria as well as microscopic fungi. Its main drawback is its instability, which makes it a logical necessity that it is to be prepared prior to use from its components HCOOH and H2O2. The mixing of 8 ml HCOOH of the concentration 850 ml/l and 17 ml H2O2 of the concentration 300 ml/l in a 100 ml-volume reagent bottle with a ground-in glass stopper gives, after an 1-hour rest at room temperature and after another 1 hour in a refrigerator, a stock solution that contains about 50 ml/l of PFA the actual concentration of which is determined iodometrically. Bacteriophage phi X 174 (host E. coli C) is characterized by cubic ikosahedral-type symmetry of particles free of envelope, has 27 mm in diameter and contains single-strand cyclic DNA; formerly was classed among Parvoviridae. The possibility of plaque assay-based quantitative determination of the number of infectious particles makes if it a feasible model for assessing disinfectant action on small hydrophilic viruses under conditions close to those of practical disinfection procedures. PFA stock solution diluted to 1 X 10(-3) (0.05 ml/l of effective component) inactivates the model virus of a concentration 10(8) pfu/ml aqueous suspension within 5 min so that no virus is detectable; the drop in the number of pfu amounts to 7 log orders of magnitude. In the presence of 400 ml/l of serum, the identical effect is achieved within 5 min by PFA stock solution diluted to 5 X 10(-3). The lowest PFA concentration that reliably inactivates bacteriophage phi X 174 in aqueous suspension is identical with the lowest concentration inactivating Coxsackie B 1 virus in tissue cultures. On textile, glass, plastic, rubber and metal carriers contaminated by swabbing or by a dried drop of bacteriophage suspension containing about 1 X 10(9) pfu/ml, the lowest reliably effective concentrations of PFA range within 0.25-0.025 ml/l, i.e. PFA stock solutions diluted to 5 X 10(-3)-5 X 10(-4), depending on the type of carrier and the type of contamination.

Bacteriophage phi X 174↗
PubMed1987

Impaired in vivo and in vitro antibody responses to bacteriophage phi X 174 in juvenile rheumatoid arthritis.

Immunologic abnormalities described in juvenile rheumatoid arthritis (JRA) have been largely confined to mitogen or antigen driven proliferation assays. We studied antigen specific antibody production using the neoantigen bacteriophage phi X 174 in vivo and in vitro; defective responses were found in all 8 patients with JRA studied. These could not be attributed to circulating anti-T cell antibodies, but may relate to lymphocyte subset abnormalities found by 2 color analyses. These immunologic aberrations may play a direct role in the pathogenesis of JRA or, alternatively, may be a secondary event.

Adolescent↗
PubMed1989

Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody.

We studied the appearance of antigen-specific immunoglobulin classes and IgG subclasses in normal adult human subjects in response to primary, secondary, and tertiary immunization with the T-cell-dependent neo-antigen bacteriophage phi X 174. To complete the study we developed a sensitive, specific, and reproducible ELISA assay which was closely comparable to the widely used neutralization assay for total antibody (r = +0.97) and for IgG antibody (r = +0.93), and reasonably comparable for IgM antibody (r = +0.76). We confirmed that the initial response to primary immunization was predominantly, but not exclusively, IgM antibody. The secondary and tertiary responses demonstrated memory, amplification, and switch from IgM to IgG antibody. There was an orderly appearance of phage-specific IgG subclasses. IgG3 and IgG1 antibodies appeared 2 to 6 weeks after primary immunization. In all subjects there was a marked increase in IgG1 and IgG3 antibody after secondary immunization, and IgG2 antibody followed closely; IgG4 antibody appeared in some subjects. IgM antibody persisted in significant amounts (approx 50%) throughout the secondary response period. Following tertiary immunization, IgG1, IgG2, and IgG3 antibody consistently increased, and IgG4 antibody appeared in all subjects; IgG1 antibody predominated. Low levels of IgM antibody (approx 1% of total) persisted during the tertiary response. The persisting antibody on long-term follow-up (median 4 years after immunization) was virtually all (greater than 90%) IgG1.

Adult↗
PubMed1977

Functions of gene C and gene D products of bacteriophage phi X 174.

Phage-related materials existing in cells infected with various mutants of bacteriophage phi chi 174 were investigated. A novel species of replicative-form (RF) DNA was found in cells infected with a phage mutant of gene B, C, D, F, or G. This species, called RFI, sedimented at a position between RFI and RFII in a neutral sucrose gradient. It was converted to RFI upon denaturation in alkali, denaturation in formamide and subsequent renaturation, or RNase treatment at low ionic strength. In cells infected with a phage mutant of gene C, RFI was derived from pulse-labeled RFII after a short chase. TLLS INFECTED WITH A MUTANT OF GENE B, D, or F. A possible function of the C gene product of phi chi 174 could be to prevent the conversion of RFII to RFI, thereby maintaining the availability of RFII to act as the template for single-stranded viral DNA synthesis. A protein complex containing no DNA, which sedimented with an S value of 108 in a sucrose gradient and contained virion proteins F, G, and H, and nonvirion protein D, was found in cells infected with the gene C mutant. A possible function of protein D was considered as a scaffolding protein for assembly of phage structural proteins.

Capsid↗
PubMed1977

Two infectious forms of bacteriophage phi X 174.

Infectious particles with S values of 114 and 132 were isolated from cells infected with bacteriophage phi chi 174. Electron micrographs of the 132S particle revealed a spherical structure with a diameter of about 40 nm. The 114S particle had spikelike projections and a diameter of about 32 nm. The 132S particles could be converted to 114S particles in vitro. However, pulse and pulse-chase experiments indicated no precursor-product relationship between these two particles in vivo.

Coliphages↗
PubMed1984

[The type of covalent bond in a natural complex of the bacteriophage phi X 174 protein A* and nucleotides].

The 32P-labelled A* protein has been isolated from E. coli cells infected by phage phi X174 in the presence of [32P]orthophosphate. The snake venom phosphodiesterase treatment of the [32P]peptides obtained by the pronase digestion of the protein has revealed a phosphodiester bond between the protein and a nucleotide material of A, G base composition. The hydrolysis of nucleotide-peptides with a mixture of concentrated HCl and CF3COOH has yielded 4'O-phosphotyrosine.

Bacteriophage phi X 174↗