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.     

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.     

Process of Infection with Bacteriophage φX174: XXXIV. Kinetics of the Attachment and Eclipse Steps of the Infection

The products of phiX cistrons II, III, and VII are demonstrated to affect the attachment of the phage to its host Escherichia coli C; therefore, by inference, these cistrons influence, directly or indirectly, the structure of proteins in the virus particle. Two of the mutations which alter attachment kinetics, ts79 in cistron III and h in cistron VII, also affect the electrophoretic mobility of the virus and emphasize the role of charge in the attachment interaction with the host. The kinetics for attached phage to go into "eclipse" are first-order and biphasic; about 85% of the phage eclipse at one rate (k(e) = 0.86 min(-1)) and the remainder do so at a distinctly lower rate (k(e) = 0.21 min(-1)). No phiX cistrons yet identified affect the eclipse process. The lowest temperature at which eclipse is detected is 19 C. The Arrhenius activation energy for phage eclipse has the high value of 36.6 kcal/mole, indicating the cooperative nature of the event.     

Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

(32)P-labeled single-stranded DNA phage phiX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in phiX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration.     

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.     

Process of Infection with Bacteriophage φX174: XXXV. Cistron VIII

Twenty-two new amber and ochre mutants of phiX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of "leakage," and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.     

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.     

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.     

Inhibition of Bacteriophage φX174 DNA Replication in dnaB Mutants of Escherichia coli C

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaE Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaE (DNA polymerase III) mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. The synthesis of the parental replicative form is unaffected, but the replication of the replicative form and the synthesis of the single-stranded virus DNA are inhibited.     

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.     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaC Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaC mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. Parental replicative form synthesis is not temperature sensitive. Single-stranded virus DNA continues to be synthesized for at least 45 min after shifting to the nonpermissive temperature late in infection. In contrast, the replication of the replicative form terminates within 5 min after shifting to the nonpermissive temperature.     

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.     

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.     

Process of Infection with Bacteriophage φX174: XXIV. New Type of Temperature-sensitive Mutant

A group of temperature-sensitive mutants of phiX174 has been isolated which can go through a single, normal one-step growth cycle at 40 C but fail to form plaques at this temperature. Such mutants fail to initiate a second cycle at 40 C; however they can gain the capacity to infect at 40 C, upon incubation for 10 min in broth at 30 C. In regaining the ability to infect, the phage appear to undergo a temperature-dependent conformational alteration. The inverse process, a reversible loss of ability to infect at 40 C, is observed when such phage produced at 30 C are incubated for 2 hr at 40 C. The defect in initiation of a second cycle of infection appears to be in the injection of viral deoxyribonucleic acid. A two-step complementation test has been used to identify the cistron coding for the affected function. Such mutants are also unusually sensitive to an irreversible thermal inactivation when incubated at 40 C.     

Process of Infection with Bacteriophage φX174: XXXVI. Measurement of Virus-Specific Proteins During a Normal Cycle of Infection

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.