Isolation and investigation of the replicating function in the coding chain of the replicative form of the DNA of phage phi-X-174.
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Coliphages↗
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phi X174-directed DNA and protein syntheses in infected minicells.
Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.
Bacteriophage phi X 174↗
Growth of a capsid mutant of bacteriophage phi X174 in a temperature-sensitive strain of Escherichia coli.
A capsid mutant of phiX174 is capable of forming replicative form and synthesizing single strands at the restrictive temperature in a dnaB mutant of Escherichia coli. Under similar conditions, the wild-type bacteriophage is incapable of either step in viral synthesis.
Carbon Radioisotopes↗
Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis.
Mutations produced in Escherichia coli by apurinic sites are believed to arise via SOS-assisted translesion replication. Analysis of replication products synthesized on depurinated single-stranded DNA by DNA polymerase III holoenzyme revealed that apurinic sites frequently blocked in vitro replication. Bypass frequency of an apurinic site was estimated to be 10-15%. Direct evidence for replicative bypass was obtained in a complete single-stranded----replicative form replication system containing DNA polymerase III holoenzyme, single-stranded DNA binding protein, DNA polymerase I, and DNa ligase, by demonstrating the sensitivity of fully replicated products to the apurinic endonuclease activity of E. coli exonuclease III. Termination at apurinic sites, like termination at pyrimidine photodimers, involved dissociation of the polymerase from the blocked termini, followed by initiations at available primer templates. When no regular primer templates were available, the polymerase underwent repeated cycles of dissociation and rebinding at the blocked termini and, while bound, carried out multiple polymerization-excision reactions opposite the apurinic sites, leading to turnover of dNTPs into dNMPs. From the in vitro turnover rates, we could predict with striking accuracy the specificity of apurinic site mutagenesis, as determined in vivo in depurinated single-stranded DNA from an M13-lac hybrid phage. This finding is consistent with the view that DNA polymerase III holoenzyme carries out the mutagenic "misinsertion" step during apurinic site mutagenesis in vivo and that the specificity of the process is determined primarily by the polymerase. SOS-induced proteins such as UmuD/C might act as processivity-like factors to stabilize the polymerase-DNA complex, thus increasing the efficiency of the next stage of past-lesion polymerization required to complete the bypass reaction.
Apurinic Acid↗
Poliovirus single-stranded RNA and double-stranded RNA: differential infectivity in enucleate cells.
The ability of poliovirus virion RNA and double-stranded RNA (replicative form) to replicate in enucleate mouse L cells was investigated. Virion RNA replicated successfully in the absence of the cell nucleus, whereas replicative form infection did not produce any detectable progeny in enucleate cells. The results provide direct evidence of a nuclear requirement early in the infection initiated by replicative form RNA.
Cell Nucleus↗
Bacteriophage phi X174 DNA synthesis in Escherichia coli HF4704S (dnaHts) cells.
The DNA synthesis of bacteriophage phiX174 in Escherichia coli HF470S, a mutant temperature sensitive in the initiation of DNA replication (dnaHts), has been examined. In HF4704S cells, phiX174 can grow normally at 27 degree C whereas the phage cannot grow after the cessation of DNA synthesis of the host cells at 42 degrees C. Upon infection, phiX174 DNA can be injected into the host cell and the parental replicative form can be formed, but the progency replicative form cannot be synthesized at 43 degrees C in the absence of host DNA synthesis. The progency replicative form cannot be synthesized at 27 degrees C in the presence of 30 mug chloramphenicol/ml in the host cell which has been incubated for 74 min at 43 degrees C followed by transfer to 27 degrees C in the presence of 30 mug chloramphenicol/ml. When 30 mug chloramphenicol/ml is added later than 5 min after the temperature shift-down to 27 degrees C, the progency replicative form synthesis is not inhibited. Thus, the host cell function, for which the gene dnaH is responsible, has been shown to be essential to the progency replicative form production.
Chloramphenicol↗
Analysis of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis.
Viral ribonucleic acid (RNA) from Semliki Forest virus- and Sindbis virus-infected cells was analyzed by electrophoresis on polyacrylamide gels. In contrast to earlier results obtained by sucrose density gradient centrifugation, all of the known viral RNA forms (i.e., the 42S, 26S, replicative form, and replicative intermediate) were very clearly separated. The high resolution of the electrophoretic method permitted the identification of two new single-stranded RNA species. In addition, the replicative form was shown to be heterogeneous and to consist of at least two forms. The results suggested that the replicative forms occur in vivo although in relatively small amounts.
Adenosine↗
Architecture of replication compartments formed during Epstein-Barr virus lytic replication.
Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.
Antigens, Viral↗
Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.
Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicative-form replication. The data suggest that these higher-than-normal-molecular-weight DNA molecules result from abnormal initiation of phiX174 replicative-form DNA replication.
Centrifugation, Density Gradient↗
Replication of M-13 DNA in plamolysed Escherichia coli cells. Formation of fully synthetic duplex DNA.
The replication of the double-stranded replicative-form DNA of bacteriophage M-13 was studied in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Newly synthesized DNA was density-labelled with bromodeoxyuridine triphosphate and analysed by equilibrium centrifugation in neutral CSCL. After a 60-min incubation at 30 degrees C 15-20% of the radioactive label in corporated from (32P)DGTP was found in fully synthetic duplex DNA, corresponding to 7-9 replicative form molecules/cell. The plasmolysed cell system is therefore capable of re-initiating new rounds of replicative form replication in vitro. The kinetics of labelling indicate that molecules are selected for replication at random from an intracellular pool of approximately 150 replicative form molecules. Rifampicin and nalidixic acid, which interfere with the semiconservative replication of replicative form DNA, completely prevent the formation of fully synthetic duplex DNA.
Bromodeoxyuridine↗
A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.
The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.
Adenosine Triphosphatases↗
On the nucleotide sequence recognized by a eukaryotic site-specific endonuclease, Endo.SceI from yeast.
Endo.SceI which is isolated from cells of Saccharomyces cerevisiae is a eukaryotic site-specific endonuclease active on double-stranded DNA. At each cleavage site, Endo.SceI cuts only a defined phosphodiester bond in each strand of the double helix. We compared nucleotide sequences around five cleavage sites for Endo.SceI using a computer. We could not find any common specific sequence consisting of five base pairs or more among them. However, we found a 26-base pair consensus sequence which included 15 conserved nucleotides, allowing any of the five sequences to include a few nucleotides deviated from the consensus sequence. The consensus sequence is 5'-CAn*PYnnAnnCYYGTTnnnPnYnnYA-3', where P, Y, n, and * denote purine, pyrimidine, any nucleotide, and the center of the cleavage site, respectively. The numbers of sites at which the consensus sequence appears in pBR322 DNA, phi X174 replicative form DNA, fd replicative form DNA, or SV40 DNA are close to those of the cleavage sites for Endo.SceI. We found that a 33-base pair fragment was efficiently cut at the defined phosphodiester bonds by Endo.SceI. This 33-base pair fragment included 25 base pairs out of the 26-base pair consensus sequence. The fragments in which a part of the consensus sequence was missing were not cut by Endo.SceI. These observations suggest that the consensus sequence described above is the major characteristic around the cleavage sites recognized by Endo.SceI and that the mode of recognition of cleavage sites by Endo.SceI is different from that by restriction endonucleases. We found homology between the consensus sequence for Endo.SceI and the sequences around the cleavage sites for two other site-specific endonucleases of S. cerevisiae: Endo.SceII and YZ-Endo which is involved in mating type switching.
Base Composition↗
A coat protein of the bacteriophage M13 virion participates in membrane-oriented synthesis of DNA.
Several molecules of a protein specified by gene 3 of M13 comprise a minor fraction of the phage coat and have been assigned a role in adsorption to the bacterial cell. We find that the gene-3 protein molecules of the virion are fully conserved in phage that have attached irreversibly to the host cell, and they form a complex with the phage DNA when it has been converted to a duplex replicative form. In cells infected at a restrictive temperature with a thermosensitive mutant in gene 3, there is no conversion of the phage DNA to the replicative form. Both the adsorbed phage and the complex of replicative form DNA with the gene-3 protein were isolated with the inner membrane fraction of the cell. We suggest that the gene-3 protein may perform an essential function in the synthesis of replicative form by linking the phage DNA to a cellular replicative system in or on the inner cell membrane.
Cell Membrane↗
Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection.
Hepatitis B virus deoxyribonucleic acid (HBV-DNA) was studied by Southern blot analysis in liver biopsy specimens from 75 HBsAg-positive patients with chronic liver disease living in southern Italy. Twenty-seven of the patients were hepatitis delta virus (HDV) superinfected. Intrahepatic HBV-DNA was detected in 54 (72%) patients, 32 (59%) of them with replicative forms. The presence of replicative forms was directly related to liver HBcAg and inversely related to liver HDAg, as shown by multivariate analysis. However, 14 patients with intrahepatic HBV-DNA non-replicative pattern and about half of HDV-infected patients were liver HBcAg and/or serum HBV-DNA positive, mostly in low amounts. Histological inflammatory activity was strongly related to liver HBcAg expression regardless of HDV superinfection, as confirmed by multivariate analysis. Our results confirm previous studies about the concordance between intrahepatic HBV-DNA replicative pattern and liver HBcAg expression and about inhibition by HDV of high-level HBV replication. However, they suggest that low-level HBV replication may have an important role in causing liver damage also among HDV-infected patients, in a population where the spreading of HBV and HDV is a naturally occurring event.
Adolescent↗
Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in lizards.
Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.
Animals↗
Superinfection in bacteriophage S13 and determination of the number of bacteriophage particles which can function in an infected cell.
Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage phiX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.
Binding Sites↗
Molecular characterization and dynamics of hepatitis C virus replication in human fetal hepatocytes infected in vitro.
The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.
Biomarkers↗
Characterization of rubella virus replication complexes using antibodies to double-stranded RNA.
A feature of the rubella virus (RV) replication cycle is the formation of cytoplasmic vesicle-containing structures known as replication complexes. Following detergent treatment of RV-infected cells, pre-embedding immunogold labeling electron microscopy using antiserum to double-stranded (ds) RNA was employed to characterize the replication complexes. Concentrations of gold particles were found associated with amorphous material located within the RV replication complex. Unlabeled long fine strands, 3-5 nm in width, were also frequently seen associated with this gold-labeled material. On some occasions gold-labeled vesicles within the replication complexes were also detected. The gold-labeled amorphous material was first detected in RV replication complexes at 12 hr postinfection, soon after the reported latent period of 8 hr. Concentrations of gold particles were not detected in mock-infected cells. The findings in this study indicate that the amorphous material is released from detergent-disrupted vesicles within the replication complex and that the vesicles contain the dsRNA. When cells were infected with the related Semliki Forest virus (SFV) and examined using the same antibody, similar gold-labeled material associated with unlabeled fine strands was also observed in SFV replication complexes. For both RV and SFV, the vesicles which line the inner membrane of the replication complexes contain the dsRNA which represent the viral replicative forms and replicative intermediates.
Animals↗