Defined oligomeric SV40 DNA: A sensitive probe of general recombination in somatic cells

We have constructed well defined oligomeric molecules of simian virus 40 (SV40) DNA as probes for investigating mechanisms by which cultured somatic cells recombine DNA. Restriction enzyme fragments from different temperature-sensitive mutants were joined in a head-to-tail orientation to create partial dimers 1.84 genome lengths in size. These molecules are too large to fit into a viral capsid. Therefore an assay that depends on production of progeny virus after infection with oligomeric DNA is a selective measure of precise conversion of oligomers to monomers. By constructing oligomers from appropriate combinations of temperature-sensitive DNAs, we have been able to study the conversion process in several defined regions of the SV40 genome. Our results indicate that conversion of oligomers to monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These data indicate that conversion occurs primarily by general, homology-dependent recombination. At least one secondary mechanism that generates a low level of wild-type progeny was also detected. Studies with heteroduplex molecules indicate that repair of mismatched bases may be the secondary mechanism.     

Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis.

The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil of [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons.     

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.     

Anatomy of Herpes Simplex Virus DNA. VIII. Properties of the Replicating DNA

This paper concerns the properties of herpes simplex virus 1 DNA replicating in HEp-2 and human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral DNA entered the replicative pool. The bulk of the input viral DNA cosedimented with marker viral DNA and did not appear to be degraded or dissociated into L and S components. (ii) Nascent DNA sedimented faster and banded at a higher density than that of mature viral DNA extracted from virions. Pulse-chase experiments indicated that nascent DNA acquires the sedimentation rate and buoyant density of viral DNA within 30 to 40 min after its synthesis. (iii) Electron microscopic studies indicated that the DNA extracted from cells replicating viral DNA and banding at the density of viral DNA contained: (a) linear, full-size molecules with internal gaps and single-stranded regions at termini; (b) molecules with lariats, consisting of a linear segment up to 2x the size of mature DNA and a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous and discontinuous forks; (c) circular, double-stranded molecules, both full-size and multiples of 18 x 10(6) in molecular weight, but without forks or loops; (d) molecules showing "eye" and "D" loops at or near one end of the DNA; (e) large, tangled masses of DNA, similar to those observed for T4 and pseudorabies virus replicating DNAs, containing loops and continuous and discontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stranded ends on the DNA anneal to form a hairpin, that the DNA synthesis is initiated at or near that end and proceeds bidirectionally to form a lariat, and that resulting progeny derived by semiconservative replication are "head-to-head" and "tail-to-tail" dimers.     

Mechanism of Replication of φX174 Single-Stranded DNA IX. Requirement for the Escherichia coli dnaG Protein

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.     

Poliovirus RNA recombination: mechanistic studies in the absence of selection.

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Mitochondrial genetics

Summary Cytoplasmic petite mutants of Saccharomyces cerevisiae carrying the gene conferring the resistance to chloramphenicol on one hand and the gene conferring the resistance to erythromycin on the other, have been crossed with each other. The two types of petites differed in the buoyant densities of their mitochondrial DNA. A novel type of evidence has been adduced, that the two genes are indeed located on mitochondrial DNA. Diploid petite recombinants were found, carrying both genes and containing not a mixture of the two parental DNAs but a new species of mitochondrial DNA of intermediate buoyant density. Recombination of mitochondrial genes involves therefore breakage and reunion of DNA molecules. New suppressiveness, different from the two parental ones, can result from the recombination of mitochondrial DNA. Recombination between petite mutants implies that the mitochondrial recombination enzymes have to be synthesized on cytosol ribosomes.     

Degradation of intracellular DNA in KB cells infected with cyt mutants of human adenovirus type 12.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.     

Requirement for Uracil-DNA Glycosylase during the Transition to Late-Phase Cytomegalovirus DNA Replication

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.     

Replication Process of the Parvovirus H-1 II. Isolation and Characterization of H-1 Replicative Form DNA

The Parvovirus H-1 replicates autonomously in hamster embryo cells. A DNA synthetic event, called HA-DNA synthesis, upon which subsequent viral RNA and viral hemagglutinin synthesis is dependent, is initiated in late S phase of the infected cell (18). It was postulated that HA-DNA represents parental viral replicative form DNA (RF DNA). This study describes the isolation and characterization of H-1 RF DNA as part of the continuing study of the mechanisms and control of DNA replication in the eukaryotic cell. The H-1 RF DNA is a linear duplex molecule containing the viral strand and its complement. The complementary strands of the RF DNA have been separated by equilibrium density gradient centrifugation. The RF DNA has a buoyant density of 1.705 in neutral CsCl and an estimated guanine plus cytosine (GC) content of 45.9%. It has a sedimentation coefficient of 17S. The calculated molecular weight of 3.7 x 10(6) is twice that of the single-stranded virion DNA. H-1 virions contain DNA that is homogeneous and free of complementary strands.     

Presence of protein at the termini of intracellular adenovirus type 5 DNA.

Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracellular viral DNA was isolated from the nuclei by lysis in 4 M guanidine hydrochloride. Electrophoresis on agarose gels of HsuI restriction enzyme fragments and sucrose gradient centrifugation were used to detect protein on intracellular viral DNA. After uncoating parental DNA still contains protein attached to the termini of the viral genome. Replicating and mature progeny viral DNA can also be isolated in the form of DNA-protein complexes. These complexes exhibit the same properties as the DNA-protein complex isolated from purified virions. These results suggest that the protein at the termini of intracellular viral DNA is identical to the protein attached to the 5'-ends of the DNA extracted from virions and that it is possibly involved in the replication of viral DNA.     

Effect of CD4 gene expression on adenovirus replication.

The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.     

Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.     

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.     

Increased infectivity of extracellular adenovirus type 12.

Human adenovirus type 12 was propagated on human embryonic kidney cells, and the specific infectivities of intra- and extracellular virus particles were compared between 48 and 104 h after infection. Released virions exhibited a specific infectivity of up to 10 times higher than that of intracellular particles. The increased infectivity was apparently not due to enhanced rates of adsorption or penetration of extracellular virus. There may be a delay in the onset of viral DNA replication in intracellular virus-infected cells. Differences in the composition of intra- and extracellular virions were not recognized. Differences might also be sought in late expression or assembly of progeny virions or both. The data indicated that the virions released from the infected cells differed from those retained in the nucleus with respect to their specific infectivities. Active mechanisms of virus release have not yet been investigated.