Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis.

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.     

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.     

Studies on the role of the phi X174 gene A protein in phi X viral strand synthesis. I. Replication of DNA containing an alteration in position 1 of the 30-nucleotide icosahedral bacteriophage origin

The influence of a C----G transversion at position 1 of the 30-base pair replication origin of bacteriophage phi X174 replicative form I DNA (phi X RFI) was examined in the RF----single-stranded circular DNA replication pathway catalyzed by the combined action of the purified phi X A protein, the Escherichia coli DNA polymerase III holoenzyme, rep helicase, and single-stranded DNA binding protein (Eisenberg, S., Scott, J.F., and Kornberg, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1594-1597; Reinberg, D., Zipursky, S.L., and Hurwitz, J. (1981) J. Biol. Chem. 256, 13143-13151). RFI DNA containing this transversion was cleaved to RFII by the phi X A protein as effectively as DNA containing the wild-type origin. The altered duplex DNA, however, supported replication at a slower rate (3- to 4-fold) than the wild-type DNA due to a defect in the termination and reinitiation reactions catalyzed by the phi X A protein. This defect resulted in the accumulation of DNA products containing long single strands covalently joined to the mutant DNA. These single strands were susceptible to nuclease S1 and exonuclease VII attack. The defect in the template DNA containing C----G transversion was not corrected when this mutant origin was placed on the same strand with a wild-type origin. This double-origin DNA was also replicated poorly and led to the accumulation of large products, in contrast to the products formed with RFI DNA containing two wild-type 30-base pair replication origins on the same strand.     

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Studies on the biological role of DNA methylation: III Role in excision of one-genome long single-stranded phi X 174 DNA.

Accumulation of replicative intermediates of the bacteriophage phi X174 was observed in E. coli C infected cells when phage DNA methylation has been inhibited by nicotinamide or when cells were infected with a temperature-sensitive mutant in gene A. Analysis of the accumulating replicative intermediates by electron microscopy revealed that these molecules are composed of double-stranded DNA rings with multiple-genome length single-stranded "tails". These results suggest that the single 5-methylcytosine residue present in the phage DNA serves as a recognition site for the gene A protein mediating the excision of one-genome long phage DNA. This excision process is oligatory for the final maturation of the phage.     

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.     

Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage phi X174.

A DNA protein complex that is a precursor of mature phi X174 phage was isolated. The complex sedimented with an S value of 50 in a sucrose gradient and contained phage DNA consisting of a replicative form molecule with an extended tail of single-stranded viral DNA. The viral-strand DNA ranged from one to two genomes in length. Proteins coded on the phi X174 genome as well as the host genome were associated with the viral DNA in the 50S precursor complex. Our results indicated that both viral DNA synthesis and cleavage of the growing viral-strand DNA occurred in the 50S complex.     

Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.     

In Vitro system for the study of bacteriophage phi chi 174 adsorption and eclipse.

An in vitro system was developed for the study of the initial stages of bacteriophage phi chi 174 infection. Escherichia coli C cells were incubated with 20% sucrose and then subjected to cold osmotic shock in 5 mM MgSO4. The concentrated supernatant shock fluid inactivated phi chi 174 with the same kinetics and requirements as for normal infection. Shock fluids prepared from phi chi 174-resistant strains of E. coli did not show this effect. The 114S phage were initially converted into 70S particles, the process termed "eclipse". These structurally altered phages then attached to a component of the shock fluid, producing fast-sedimenting complexes, and eventually released at least a part of their DNA into the medium. The fast-sedimenting complex could be radioactively labeled with oleic acid. Radioactivity was found to co-chromatograph with both biological activity and the majority of the high-molecular-weight carbohydrates present in the shock fluid. It is concluded that E. coli C osmotic shock fluid contains isolated phi chi 174-specific receptor sites composed of lipopolysaccharides. This system conveniently separates the early stages of phage phi chi 174 infection from the intracellular events.     

Construction and properties of a ribosome-binding site mutation in gene E of phi X174 bacteriophage.

Oligodeoxyribonucleotide mutagenesis has been used to produce a G----A mutation at nucleotide 557 of the phi X174 genome. This changes the ribosome-binding sequence GAGG of gene E to GAAG without affecting the amino acid, glutamine, encoded by the overlapping gene D. The phi X174rb(E)557 mutant does not lyse infected Escherichia coli C and therefore results in the accumulation of a large number intracellular mature phage particles. Thus, the mutation inactivates production of the gene E lytic product, presumably by blocking translation of gene E, without affecting other phage functions.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Role of polymeric forms of the bacteriophage phi X174 coded gene A protein in phi XRFI DNA cleavage.

Gene A of the phi X174 genome codes for two proteins, A and A* (Linney, E.A., and Hayashi, M.N. (1973) Nature New Biol. 245, 6-8) of molecular weights 60,000 and 35,000, respectively. The phi X A* protein is formed from a natural internal initiator site within the A gene cistron while the phi X A protein is the product of the entire A gene. These two proteins have been purified to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Previous studies have shown that the phi X A protein is an endonuclease which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted phi XRFI DNA. In addition to this activity, the phi X A protein also causes relaxation of supertwisted phi XRFI DNA and formation of a phi XRFH DNA . phi X A protein complex which has a discontinuity in the A cistron of the viral strand. This isolatable complex supports DNA synthesis when supplemented with extracts of uninfected Escherichia coli which lack phi X A protein and phi XRFI DNA. The phi XRFII DNA . phi X A protein complex can be attacked by exonuclease III but is not susceptible to attack by E. coli DNA polymerase I, indicating that the 5'-end of the complex is blocked. Attempts to seal the RFII structure generated from the phi XRFII DNA . phi X A protein complex with T4 DNA ligase in the presence or absence of DNA polymerase were unsuccessful. The phi X A protein does not act catalytically in the cleavage of phi XRFI DNA. Under conditions leading to the quantitative cleavage of phi XRFI DNA, the molar ratio of phi XRFI DNA to added phi X A protein was approximately 1:10. At this molar ratio, cross-linking experiments with dimethyl suberimidate yielded 10 distinct protein bands which were multiples of the monomeric phi X A protein. In the absence of DNA or in the presence of inactive DNA (phi XRFII DNA) no distinct protein bands above a trimer were detected. We found it possible in vitro to form a phi XRFII DNA . phi X A protein complex with wild-type phi XRFI DNA (phi X A gene+) and with phi XRFI DNA isolated from E. coli (su+) infected with phage phi X H90 (an am mutant in the phi X A gene). Thus, in vitro, in contrast to in vivo studies, phi X A protein is not a cis acting protein. The purified phi X A* protein does not substitute for the phi X A protein in in vitro replication of phi XRFI DNA nor does it interfere with the action of the phi X A protein which binds only to supertwisted phi XRFI DNA. In contrast, the phi X A* protein binds to all duplex DNA preparations tested. This property prevents nucleases of E. coli from hydrolyzing duplex DNAs to small molecular weight products.     

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.     

Gene A product of phi X174 is required for site-specific endonucleolytic cleavage during single-stranded DNA synthesis in vivo.

A functional gene A product of phi X174 was found to be required at the stage of single-stranded DNA synthesis. A precursor complex that synthesizes single-stranded DNA (50S complex [Fujisawa and Hayashi, 1976]) was isolated from cells infected with wild-type or with temperature-sensitive gene A mutant phage. Proper cleavage of the single-stranded viral DNA did not occur in cells infected with the temperature-sensitive gene A mutant under restrictive conditions. This resulted in (i) accumulation of linear viral DNA molecules of 2 units in length in the 50S complex and (ii) cessation of elongation of viral-strand DNA after one complete cycle of single-stranded DNA synthesis.     

Growth and DNA synthesis of bacteriophage phi x174 in a dnaP mutant of Escherichia coli.

phi X174am3trD, a temperature-resistant mutant of bacteriophage phi X174am3, exhibited a reduced ability to grow in a dnaP mutant, Escherichia coli KM107, at the restrictive temperature (43 degrees C). Under conditions at which the dnaP gene function was inactivated, the amount and the rate of phi X174am3trD DNA synthesis were reduced. The efficiency of phage attachment to E. coli KM107 at 43 degrees C was the same as to the parental strain, E. coli KD4301, but phage eclipse and phage DNA penetration were inhibited in E. coli KM107 at 43 degrees C. It is suggested that the dnaP gene product, which is necessary for the initiation of host DNA replication, participates in the conversion of attached phages to eclipsed particles and in phage DNA penetration in vivo in normal infection.     

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III.

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.     

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.     

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.     

φX-174 Bacteriophage Structural Mutants Which Affect Deoxyribonucleic Acid Synthesis

Seven cistrons in X-174 were identified and one in particular was studied intensively: cistron A, which is assigned a protein in the mature phage. Amber mutants in this cistron synthesize a new deoxyribonucleic acid (DNA) form in addition to circular phage DNA upon infection of the restrictive host. This DNA is linear, non-infectious, and single-stranded; it is formed from the phage strand of replicative form X-174 DNA. These mutants produce two different defective particles in the restrictive host. One particle contains circular phage DNA but is not infectious; the other contains the new DNA form and is similar to the 70S particles found in wild-type phage lysates. The mutant A gene product acts independently of normal A protein upon mixed infection of the restrictive host with an A mutant and a mutant from any other cistron or wild type.