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.     

DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns.

A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

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.     

Loss of supercoiled viral DNA after infection of permissive cells by simian virus 40.

During infection of BS-C-1 cells (a permissive line of monkey kidney cells) with simian virus 40, there was a loss of infecting viral supercoiled (component I) DNA molecules which was partially inhibited by cycloheximide. The kinetics of loss of component I in the presence of cycloheximide suggested that the loss was the result of at least two activities acting on the DNA, one cycloheximide sensitive and the second cycloheximide insensitive. Nucleotides from degraded infecting viral DNA were reincorporated into cell DNA that was synthesized during the infection.     

Inhibition of protein synthesis enhances transformation of primary cells by viral DNA

Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK-) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency.     

Covalently closed circles of human adenovirus DNA are infectious

Replication of the linear adenovirus DNA molecule is thought to result from semiconservative synthesis off linear templates, starting from origins at either end of the genome. Recently, however, it has been shown that in cells infected with adenovirus type 5 (Ad5) a significant fraction of the ends of viral DNA molecules become joined head-to-tail due at least in part to the formation of covalently closed circles. Circular DNA is not present in virions but joining of the ends of viral DNA is detectable shortly after infection, well before the onset of viral DNA replication. To learn more about the structure and possible function of these circular forms of viral DNA, I have cloned Ad5 circles as plasmids replicating in Escherichia coli. Two plasmids have been analyzed in detail and shown to generate infectious virus with an efficiency comparable with that of virion DNA following transfection into human cells. These results suggest that circles are not totally inert or functionless but that, once formed, they are capable of re-entering the pool of replicating molecules to generate linear progeny.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.     

Synthesis of multimeric polyoma virus DNA in mouse L-cells: role of the tsA1S9 gene product.

Several different forms of progeny viral DNA can be identified in polyoma virus (Py)-infected mouse L-cells. The majority comprise mature form I superhelical DNA and the circular, double-stranded "theta" replicating intermediates in which the progeny DNA strands never exceed the unit genome length of the template. There is formed, in addition, a minority fraction of multimeric, linear, double-stranded Py DNA molecules that sediment heterogeneously at 28 to 35S and greater than 35S. Restriction enzyme analysis of these large Py DNA molecules reveals them to be tandem arrays of multiple unit genome lengths, covalently linked head to tail. It is estimated that the 28 to 35S multimeric DNA has an average size of about 20 megadaltons, made up of 6 to 20 Py genome units. The greater than 35S Py DNA is, of course, larger. Kinetic analysis indicates that formation of the monomeric progeny viral DNA and the 28 to 35S multimeric Py DNA reaches a peak at about 35 to 36 h postinfection. Synthesis of the very large linear molecules of greater than 35S is first detected after this interval and continues thereafter. The de novo synthesis of all of these progeny Py DNA molecules proceeds apparently normally in Py-infected tsA1S9 mouse L-cells incubated at 38.5 degrees C under conditions which restrict normal cellular DNA replication. These findings suggest that the cellular DNA topoisomerase II activity, encoded in the tsA1S9 locus (R. W. Colwill and R. Sheinin, submitted for publication), is not required for de novo formation of any form of Py DNA. However, the total amount made and the rate of synthesis of the large molecular weight Py DNA are affected very late in temperature-inactivated tsA1S9 cells.     

Protein synthesized early after infection is linked to the termini of adenovirus type 2 DNA synthesized in vivo and in vitro.

The human adenovirus DNA genome contains a protein (CBP, or covalently bound protein) linked to each 5' terminus. To assess whether CBP is synthesized early, infected cells were incubated with hydroxyurea from 1 to 18 h postinfection, the hydroxyurea was removed, cycloheximide was added, and viral DNA was labeled with [3H]thymidine from 18 to 23 h postinfection. Removal of hydroxyurea at 18 h postinfection permits the synthesis of viral DNA, whereas cycloheximide maintains the block in late viral protein synthesis. Three lines of evidence are presented to show that viral 3H-labeled DNA prepared by this procedure was linked to CBP: (I) the DNA sedimented more rapidly than protein-free DNA (i.e., protinase treated) in neutral sucrose gradients containing guanidine hydrochloride; (ii) the DNA banded at a lower density than protein-free DNA in CsCl gradients containing guanidine hydrochloride; and (iii) neither the 3H-labeled DNA nor the end fragments produced by EcoRI digestion entered a 1.4% agarose gel during electrophoresis. These experiments are strong evidence that CBP is not a product of a late viral gene and is therefore the product of either an early viral gene or a cell gene. Experiments were performed to test whether CBP is attached to viral DNA synthesized in vitro by a soluble complex that synthesizes exclusively viral DNA as completed viral genomes in vitro. In vitro-labeled DNA was analyzed by velocity sedimentation, equilibrium sedimentation, and agarose gel electrophoresis as described above. Our results indicate that the majority of in vitro-synthesized DNA molecules were attached to CBP. These results, which indicate that CBP is synthesized early after infection and is attached to viral DNA labeled in vitro by a soluble replication complex, are consistent with the idea that CBP may play a role in viral DNA replication.     

Synthesis of the adenovirus-coded DNA binding protein in infected cells.

Synthesis of the 75K (75K indicates a moleculatr weight of 70,000 to 75,000) DNA binding protein, an early virus-coded protein in adenovirus 2-infected KB cells, and its regulation were studied by using a radioimmune precipitation inhibition assay. The protein was first detected at 4 h postinfection and accumulated at an expoential rate. An arrest of further synthesis (accumulation) was observed at 10 to 11 h postinfection, coinciding with the onset of synthesis of late virion proteins. In contrast, when the infected cells were treated with 25 mug of arabinosyl cytosine per ml to block viral DNA replication, the synthesis of 75K protein did not cease but continue for up to 36 h postinfection. The synthesis of 75K protein in cells after release from a cycloheximide block (2 to 9 h postinfection) was analyzed. Increased amounts of early adenovirus-specific mRNA accumulate in infected cells during a cycloheximide block (Parsons and Green, 1971). However, cycloheximide treatment did not produce increased levels of 75K protein, and an abrupt arrest of 75K protein formation was again observed at the time of synthesis of late virion proteins. Partition of the 75K protein between the nuclear and cytoplasmic fractions during the course of infection was studied. The 75K protein appeared first in the cytoplasm and then in the nucleus after a slight lag. Accumulation of the 75K protein continued both in the cytoplasm and nucleus, with higher levels being found in the cytoplasm.     

Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection.

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Effect of protein synthesis inhibitors on DNA replication. I. Effect of the short-term action of cycloheximide on cell permeability for thymidine in vitro]

The effect of protein synthesis inhibitors on DNA replication was studied on L cells. After a 10 minutes' action of the inhibitors, protein synthesis was seen to be completely blocked, and DNA synthesis decreased by 85%. Four hours after a 20-minutes' cycloheximide treatment, the cells completely restored their ability to protein synthesis and DNA replication and even surpass the control level, due, probably, to a partial cell synchronization in S period. The short action of cycloheximide did not interfere with thymidine uptake by the cells. The rate of the exogenous precursor uptake was even higher than that in the control, apparently, because of its much reduced utilization in the inhibited DNA synthesis.     

Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI^*) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished three- to fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80% completed SV40(RI) DNA. Failure to convert SV40(RI^*) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with their nascent DNA strands interrupted in the termination region [SV40(II^*) DNA] accumulated with unjoined Okazaki fragments. Thus, separation of sibling chromosomes still occurred, but gaps remained in the terminal portions of their daughter DNA strands. These and other data support a central role for SV40(RI^*) and SV40(II^*) DNAs in the completion of viral DNA replication.     

Inhibition of retrovirus DNA supercoiling in interferon-treated cells.

The effect of interferon (IFN) on the cytoplasmic synthesis of murine sarcoma virus DNA, its transport to the host nucleus, and its integration into the cellular genome were investigated. For this purpose, at various intervals after infection. DNA was extracted from the cytoplasmic fraction, nuclear Hirt supernatant, and chromosomal DNA pellet. The relative amount of viral DNA was estimated by C0t kinetics analysis of hybridization to murine sarcoma virus-specific [3H]cDNA. IFN was found to delay viral DNA synthesis by about 2.5 h, but the amount of viral DNA eventually formed in IFN-treated cells was comparable to that of the control. The transport of this DNA to the nucleus was delayed by IFN for 6 to 18 h, but once again, all the cytoplasmic viral DNA formed in IFN-treated cells was eventually transferred to their nucleus. However, although the main part of the viral DNA formed in control cells was finally integrated into the host genome, no significant integration was observed in IFN-treated cells. Alkaline sucrose gradient analysis revealed that IFN inhibited the accumulation of supercoiled viral DNA in the nucleus. Since supercoiled viral DNA is considered a precursor to integrated provirus, this observation may suggest that IFN inhibits viral DNA integration by blocking its supercoiling.