Effect of Ultraviolet Irradiation and Actinomycin D on Polyoma Virus Replication in Mouse Embryo Cell Cultures

Ultraviolet irradiation and actinomycin D impair the capacity of mouse embryo (ME) cells to support the replication of polyoma virus, but not of encephalomyocarditis (EMC) virus. The loss in capacity for polyoma virus synthesis was an “all-or-none” effect and followed closely upon the loss in cellular capacity for clone formation. Cells treated with either agent produced polyoma “T” antigen, but did not synthesize polyoma structural protein. Infection of untreated ME cells with polyoma virus produced marked stimulation of both deoxyribonucleic acid (DNA) synthesis and ribonucleic acid (RNA) synthesis. ME cell cultures irradiated with ultraviolet for 30 sec at 60 μw/cm² or treated with actinomycin D at 0.1 μg/ml for 6 hr prior to infection were incapable of synthesizing DNA or RNA, even after infection with polyoma virus. Irradiation of cells during infection produced cessation of synthesis of both RNA and DNA. Addition of actinomycin D during infection did not inhibit DNA synthesis but abolished RNA synthesis and reduced the yield of polyoma virus to 10% of that in untreated infected cultures. Both agents lost the ability to prevent replication of a full yield of polyoma virus when administered 30 hr after infection or later. The period after which neither agent inhibited polyoma replication corresponded with the period at which maximal RNA synthesis in untreated infected cultures had subsided. It can be concluded on the basis of the data presented that the functional integrity of the mouse embryo cell genome is required for the replication of polyoma virus, but not for EMC virus. Whereas the requirement for cellular DNA-dependent RNA synthesis for polyoma virus replication has been demonstrated, the exact nature of the host-cell function remains to be elucidated.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication I. Characterization of Fragments

Hydroxyurea treatment of 3T6 mouse fibroblast cells infected with polyoma virus resulted within 15 min in more than a 20-fold reduction of the rate of both viral and cellular DNA synthesis. After the initial rapid inhibition, the rate of DNA synthesis remained essentially constant for at least 2 h. In the inhibited cells viral DNA accumulated as short chains with a sedimentation coefficient of about 4S (hydroxyurea fragments). A variable proportion of these fragments was released from the template strands when the viral DNA was extracted by the Hirt procedure. Reannealing experiments demonstrated that hydroxyurea fragments were polyoma-specific and probably synthesized on both parental strands at the replication forks.     

Formation of Cellular Deoxyribonucleic Acid During Productive Polyoma Virus Infection

It was previously shown that the majority of deoxyribonucleic acid (DNA) made in growing mouse embryo cells productively infected at low multiplicity with polyoma virus is cellular in nature and that some of this cell DNA contains discontinuities in the newly synthesized strand. Evidence obtained indicates the following. (i) Induction of cell DNA synthesis precedes the onset of detectable viral DNA replication by approximately 3 hr. (ii) Double-stranded cell DNA molecules, discontinuous in the newly synthesized strands, arise by direct synthesis (rather than by degradation of a high-molecular-weight precursor) only in the cell DNA replicated after initiation of viral DNA synthesis. (iii) This DNA component is continuously formed throughout the "late" stage of infection and is continuously converted into apparently normal cell DNA of high molecular weight without prior degradation to acid-soluble components.     

Vitamin A inhibition of polyoma virus replication

Vitamin A (retinoic acid) inhibited polyoma virus replication in confluent mouse embryo cells. A significant, dose dependent inhibition was observed when cell monolayers were pretreated with concentrations of vitamin A (10(-8) to 10(-6) M) thought to approximate those found in vivo. This inhibitory effect could be reduced by increasing the input multiplicity of infection. Growth curves of polyoma virus in the presence and absence of vitamin A suggested that vitamin A actually inhibited, and did not simply delay, virus replication. The cell density dependence of this inhibitory effect suggested its association with the prevailing level of cellular DNA synthesis. Vitamin A caused a significant decrease in overall (viral plus cellular) DNA synthesis. Other viruses which do not require induction of host cell DNA synthesis for their replication in confluent, non-dividing cells were not inhibited by vitamin A. These results are consistent with the known inhibitory effects of vitamin A on papovavirus infection in vivo and suggest a mechanism of vitamin A action at the level of the infected cell.     

Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA.

We have used two experimental strategies to test the role of cellular functions in the synthesis and integration of virus-specific DNA in cells infected by avian sarcoma virus.First, quail embryo fibroblasts, placed in stationary phase (G₀) by prolonged serum starvation, did not support the efficient synthesis of viral DNA during the first 24–48 hr after infection. Synthesis of viral DNA was impaired according to at least two parameters: the amount of DNA was diminished, particularly the amount of the plus-strand DNA (identical in polarity to the viral genome); and the length of both minus and plus strands was reduced in the stationary cells. In parallel cultures fed with fresh serum, over two thirds of the cells were able to reenter the cell cycle within 24 hr, and viral DNA of normal size was synthesized.Second, density labeling of viral and cellular DNA with BUdR was used to determine whether cellular DNA synthesis was required for integration of viral DNA. In both quail embryo fibroblasts released from G₀ by serum replacement and randomly growing duck embryo fibroblasts, viral DNA was integrated only into cellular DNA replicated during the infection.Our results indicate that serum-starved cells lack a factor (or factors) required for the efficient and complete synthesis of ASV-specific DNA. We have not been able to establish whether such factor(s) are present in growing cells only during S phase. Integration of viral DNA appears to require cellular DNA synthesis; this may be due to a requirement for a factor (or factors) present in adequate concentration only during S phase or to a requirement for the structural changes in cellular DNA that accompany replication.     

Interactions of Polyoma and Mouse DNAs. IV. Time Course and Extent of Integration of Polyoma DNA into Mouse DNA During Lytic Infection

The time course of covalent binding of polyoma viral DNA to mouse DNA was followed in mouse embryo cells that had been grown prior to infection in the presence of 5-bromodeoxyuridine. Density-labeled (HL) mouse DNA was separated from free polyoma DNA by CsCl isopycnic centrifugation. Polyoma DNA sequences present in HL mouse DNA were detected by hybridization with radioactive cRNA synthesized in vitro. In reconstruction experiments, the limit of detection was found to be, on the average, about 0.5 genome equivalent (g.e.) of polyoma DNA per cell. To find conditions for the isolation of HL mouse DNA and for its complete separation from free polyoma DNA, cultures infected at 4 degrees C were used. HL mouse DNA extracted with sodium dodecyl sulfate and high salt concentrations (5 to 6 M CsCl) and then purified by three consecutive CsCl density gradient centrifugations was free from detectable amounts of polyoma DNA, whereas HL mouse DNA extracted with chloroform and phenol and purified in the same way always contained contaminating, noncovalently bound polyoma DNA. In lytically infected bromodeoxyuridine-prelabeled mouse embryo cultures, polyoma DNA bound to HL mouse DNA that had been extracted by the sodium dodecyl sulfate-CsCl procedure was first detected in small amounts (1 to 2 g.e. per cell) at 10 h after infection. In cultures incubated with medium containing thymidine (5 mug/ml), 4 to 6 g.e. of polyoma DNA per cell was detected at 14 and 18 h after infection. In these samples, practically all viral DNA was bound to high-molecular-weight HL mouse DNA. In cultures incubated with normal medium (no additions) and extracted between 17 and 20 h after infection, 20 to 350 g.e. of polyoma DNA per cell banded with HL mouse DNA. However, when DNA of one of these samples was subfractionated by sodium dodecyl sulfate-salt precipitation prior to isolation of HL mouse DNA, about 80% of the viral DNA banding at increased density was present in the low-molecular-weight DNA fraction. This observation suggests that in normal medium some progeny viral DNA of increased density was synthesized. Covalent binding of polyoma DNA to density-labeled mouse DNA was demonstrated by alkaline CsCl density gradient centrifugation: nearly equal amounts of polyoma DNA were found in the H and L strands, respectively, as is expected for linear integration of viral DNA. The results lead to the conclusions that (i) early polyoma mRNA is transcribed from free parental viral DNA; (ii) covalent linear integration is first detectable at the time when tumor (T)-antigen is synthesized; and (iii) only few copies (<10 g.e./cell) become integrated between 10 and 18 h after infection, i.e., during the period when cellular and viral DNA replication starts in individual cells.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

Selective antiviral agents. The metabolism of 5-propyl-2'-deoxyuridine and effects on DNA synthesis in herpes simplex virus type 1 infections

The metabolism and disposition of 5-propyl-2'-deoxyuridine (Pr-dUrd) in herpes simplex virus type 1 infections were investigated in cell culture using [14C]Pr-dUrd, [32P]orthophosphate, and several methods including high pressure liquid chromatography and isopycnic centrifugation. Results in infected cells indicate Pr-dUrd 1) is taken up and phosphorylated to mono-, di-, and triphosphates; 2) is incorporated into DNA; 3) preferentially inhibits synthesis of viral DNA; 4) blocks re-initiation of viral DNA synthesis even after removal of the nucleoside from the culture; and 5) exerts these effects early in the course of infection (before 6 h postinfection). Pr-dUrd was not phosphorylated in uninfected cells, and had little or no effect on apparent cellular DNA synthesis in infected or uninfected cells. Present evidence suggests one possible antiviral event could be the lethal effect of Pr-dUrd after incorporation into viral DNA by alteration of DNA template-directed functions such as replication.     

Further Studies on the Effect of 5-Iododeoxyuridine on Polyoma Virus Multiplication in Mouse Cells

5-Iododeoxyuridine (IUDR) inhibited production of infectious polyoma virus in mouse embryo cells and mouse kidney cells in culture. Deoxythymidine reversed its effect. IUDR did not inactivate infectivity of free virus particles. IUDR did not prevent adsorption and penetration of polyoma virus to cells. The events sensitive to IUDR treatment occurred at around 20 hours after infection. The cytopathic effects of polyoma virus, including emergence of DNA containing-inclusions in the nucleus, were observable in infected cells in which viral replication was completely arrested by IUDR. It was shown by fluorescent antibody technique in infected mouse embryo cells and by complement fixation test in infected mouse kidney cells that IUDR inhibited completely the synthesis of viral antigen. No virus-like particles were demonstrated in the IUDR-treated infected-mouse kidney cells by electron microscope examinations.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Adenovirus DNA Replication I. Requirement for Protein Synthesis and Isolation of Nuclear Membrane Fractions Containing Newly Synthesized Viral DNA and Proteins

Nuclear membrane fractions were prepared by two procedures from KB cells pulse labeled with [(3)H]thymidine for 5 min late after infection with adenovirus 2: (i) the M-band technique, which yields a sharp peak containing most of the newly synthesized viral DNA, and (ii) the discontinuous sucrose gradient method, which yields three membrane fractions, one which bands at the interface between sucrose layers at density 1.18 and 1.20 g/ml and contains most of the newly synthesized viral DNA. Studies using cycloheximide to inhibit protein synthesis showed that proteins whose synthesis begins early after infection and occurs in the absence of viral DNA replication are required for viral DNA synthesis late after infection. To study the nature of these proteins, nuclear membrane fractions were isolated from cells labeled with [(3)H]leucine from 6 to 24 h postinfection in the presence of arabinosyl cytosine to block viral DNA replication, and were analyzed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Two proteins of molecular weights 75,000 and 45,000 were the major labeled polypeptides in the nuclear membrane fractions prepared from infected cells both by the M-band and the discontinuous sucrose gradient methods. These two proteins were not found in nuclear membrane fractions from uninfected cells. It is suggested that the 75,000 and 45,000 proteins may be early viral gene products that may play a role in the viral DNA replication.     

DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication.

Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.     

Replication of Epstein–Barr Viral DNA

Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding.     

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.     

Effects of arabinosyl-cytosine on thymidine triphosphate pools and polyoma DNA replication.

Arabinosyl cytosine at very low concentrations (5–100 nmolar) inhibits the incorporation of [³H]thymidine into polyoma DNA of infected mouse fibroblasts without affecting the labeling of the [³H]dTTP pool. The specific activities of these pools were determined by a new simple method. Inhibition of DNA synthesis affects chain elongation and not initiation of new rounds of replication.     

Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.