Addition of extra DNA sequences to simian virus 40 DNA in vivo.

The possible addition of extra sequences to simian virus 40 (SV40) DNA was analyzed by electron microscopy in two different cell systems, productively infected monkey cells and activated heterokaryons on monkey and transformed mouse 3T3 cells. We found that the closed circular DNA fraction, extracted from monkey cells at 70 h after infection with nondefective SV40 at a multiplicity of infection of 6 PFU/cell, contained oversized molesules (1.1 to 2.0 fractional lengths of SV40 DNA) constituting about 8% of the molecules having lengths equal to or shorter than SV40 dinner DNA. The oversized molecules had the entired SV40 sequences. The added DNA was heterogeneous in length. The sites of addition were not specific with reference to the EcoRi site. These results suggest that recombination between monkey and SV40 DNAs or partial duplication of SV40 DNA occurs at many sites on the SV40 chromosome. The integrated SV40 DNA is excised and replicates in activated heterokaryons. In this system, besides SV40 DNA we found heterogeneous undersized and oversized molecules containing SV40 sequences in the closed circular DNA population. Additions differeing in size appeared to be overlapping and to have occurred at a preferential site on the SV40 chromosome. These results support the hypothesis that host DNA can be added to SV40 DNA at the site of integration at the time of excision.     

Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid

Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection.     

Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid

The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.     

Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

Simian Virus 40 Deoxyribonucleic Acid Replication: I. Effect of Cycloheximide on the Replication of SV40 Deoxyribonucleic Acid in Monkey Kidney Cells and in Heterokaryons of SV40-transformed and Susceptible Cells

Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-β-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with ³H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with ³H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication.     

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.     

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.     

Simian Virus 40 Deoxyribonucleic Acid Synthesis: the Viral Replicon

Three temperature-sensitive (ts) mutants of simian virus 40 (SV40) in complementation group A (tsA7, tsA28, tsA30) have been isolated and characterized in permissive and restrictive host cells. At 41 C in the AH line of African green monkey kidney cells, the mutants are deficient in an early function required to produce infectious viral deoxyribonucleic acid (DNA). Temperature-shift experiments and analysis of SV40 viral DNA replication by gel electrophoresis have provided strong evidence that the ts gene product of the three mutants is directly required to initiate each new round of viral DNA replication but is not required to complete a cycle which has already begun. The synthesis of mutant DNA molecules themselves can be initiated by a nonmutant gene product in viral complementation studies at 41 C. The cell, however, cannot substitute a host function to provide the initiator required for the replication of free viral DNA. The viral initiator is also required to establish the stable transformation of 3T3 cells.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA III. Host Sequences

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.     

Superhelix Density Heterogeneity of Intracellular Simian Virus 40 Deoxyribonucleic Acid

Covalently closed intracellular and viral simian virus 40 (SV40) deoxyribonucleic acid (DNA) were separately isolated from infected African green monkey cells (BSC-1) grown in culture. The two DNA species form overlapping bands centered at different positions in a propidium di-iodide-cesium chloride (PDI-CsCl) buoyant density gradient capable of separating closed DNA species with different superhelix densities. When the dense side of a (32)P-labeled intracellular DNA band was mixed with the light side of a (3)H-labeled intracellular DNA band and again centrifuged in a PDI-CsCl density gradient, two overlapping bands formed with modes displaced from each other. Similar band-splitting experiments performed with viral DNA always gave superimposable bands. The foregoing experiments demonstrate that the intracellular DNA is heterogeneous in superhelix density, whereas, by the same criteria, the viral DNA is homogeneous. The mean superhelix density of the intracellular closed DNA is approximately three-fourths as large as the superhelix density of the viral DNA. These results rule out the possibility that closed SV40 DNA is drawn randomly from the intracellular pool and assembled without a further nicking-closing step into virions. When the cells were grown and infected in the presence of ethidium bromide (EB), the intracellular closed DNA was found to be homogeneous in superhelix density and to have the same superhelix density as the viral DNA which, in turn, was unaffected by the presence of the drug. The foregoing results were explained by postulating that the intracellular DNA is formed with a homogeneous superhelix density and becomes heterogeneous in the absence of EB as a result of a nicking-closing cycle that occurs in a spacially or temporally heterogeneous environment. The drug EB would inhibit this action by inhibiting the nicking enzyme(s).     

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.     

Synthesis of Superhelical Simian Virus 40 Deoxyribonucleic Acid in Cell Lysates*.

In vivo-labeled SV40 replicating DNA molecules can be converted into covalently closed superhelical SV40 DNA (SV40(I) using a lysate of sv40-infected monkey cells containing intact nuclei. Replication in vitro occurred at one-third the in vivo rate for 30 min at 30 degrees. After 1 hour of incubation, about 54% of the replicating molecules had been converted to SV40(I), 5% to nicked, circular molecules (SV40(II), 5% to covalently closed dimers; the remainder failed to complete replication although 75% of the prelabeled daughter strands had been elongated to one-genome length. Density labeling in vitro showed that all replicating molecules had participated during DNA synthesis in vitro. Velocity and equilibrium sedimentation analysis of pulse-chased and labeled DNA using radioactive and density labels suggested that SV40 DNA synthesis in vitro was a continuation of normal ongoing DNA synthesis. Initiation of new rounds of SV40 DNA replication was not detectable.     

Multiplication of Polyoma Virus in Mouse-Hamster Somatic Hybrids: a Hybrid Cell Line Which Produces Viral Particles Containing Predominantly Host Deoxyribonucleic Acid

The multiplication of polyoma virus in a mouse-hamster (3T3 x BHK) somatic hybrid line (10A), which, although permissive for viral multiplication, produces very low amounts of virus, has been studied. In this cell line, the efficiency of productive infection is high, but the yield of infectious virus is on the order of 0.5% of that of 3T3 cells. The amount of viral deoxyribonucleic acid (DNA) synthesized by these cells upon infection is about 5% of that of 3T3 cells. An examination of the virus produced in hybrid 10A revealed that it was only one-tenth as infectious as the virus grown in 3T3. Although the viral DNA synthesized in the infected 10A cells is normal, the DNA extracted from purified virus grown in 10A consists of approximately 10% of normal, supercoiled polyoma DNA molecules and of approximately 90% linear DNA molecules with a sedimentation coefficient of 14 to 16S. These DNA molecules appear to be of cellular origin but contain a limited amount of viral DNA sequences. The host DNA-containing particles are not infectious but appear to possess some biological activity; they give rise to a weak complementation effect, and part of them are able to induce T-antigen synthesis. In addition, the host DNA present in these particles is predominantly that which has been synthesized after infection. The correlation between the block in viral DNA synthesis in this cell line and the abnormal encapsidation of host DNA is discussed.     

Regulation of SV40-induced cell division and tumor antigen by dibutyryl adenosine 3′-5′-monophosphate

Simian virus 40 (SV40) induces cell division in microcultures of sparsely plated nongrowing mouse BALB/3T3 cells during acute infection at moderate multiplicities of infection (MOI = 10–100). The infected cells are killed when a MOI of 1,000 is used. SV40 tumor (T) antigen is synthesized in the infected cells, but viral DNA, virion antigen, and progeny virions are not synthesized (abortive infection). The addition of exogenous dibutyryl adenosine 3′-5′-monophosphate (dbcAMP) at the time of infection stimulates the SV40-induced cell division at all MOI and inhibits SV40-induced cell death at high MOI. The percentage of T antigen-positive cells, as monitored by immunofluorescence, is also increased by the addition of dbcAMP at the time of infection. This regulation of SV40-induced cell division and T antigen formation by exogenous dbcAMP occurs within the first 6 hr after infection at 37° C and is dependent upon both the MOI and the concentration of added dbcAMP. The addition of dbcAMP to productively infected TC7 monkey cells has little effect on the SV40-induced cell death or T antigen formation.     

Requirement of a Cytoplasmic Fraction for Synthesis of SV40 Deoxyribonucleic Acid in Isolated Nuclei*.

About 50% of the SV40 DNA in the process of replication (sv40(ri) dna) completed replication in lysates of infected BSC-1 cells by conversion to covalently closed, superhelical SV40 DNA (SV40(I) DNA). Fractionation of the lysate into nuclear and cytoplasmic components blocked 99% of the synthesis of SV40(I) DNA in the purified nuclei. The reconstituted system, made by adding back the cytoplasmic fraction before incubation at 30 degrees, completely restored the in vitro level of SV40(I) DNA synthesis. Preliminary characterization of the activity found in the cytoplasmic fraction suggested it was a soluble, heat-labile protein (or proteins) with a minimum molecular weight of about 30,000 and an active sulfhydryl group. The activity was present in both infected and uninfected monkey cells, and at a lower level in mouse, hamster, and human cell lines. Neither serum starvation nor cycloheximide treatment of cells diminished the activity in the cytoplasmic fraction. Purified cytoplasmic DNA polymerase from KB cells did not substitute for the cytoplasmic fraction which was required for elongation of newly synthesized DNA strands. In the absence of the cytoplasmic fraction, conversion of 4 S DNA into longer strands was inhibited, and SV40(RI) DNA appeared to be broken specifically at the replication forks.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Deoxyribonucleic Acid Synthesis in FV-3-infected Mammalian Cells

Deoxyribonucleic acid (DNA) synthesis and virus growth in frog virus 3 (FV-3)-infected mammalian cells in suspension were examined. The kinetics of thymidine incorporation into DNA was followed by fractionating infected cells. The cell fractionation procedure separated replicating viral DNA from matured virus. Incorporation of isotope into the nuclear fraction was depressed 2 to 3 hr postinfection; this inhibition did not require protein synthesis. About 3 to 4 hr postinfection, there was an increase in thymidine incorporation into both nuclear and cytoplasmic fractions. The nuclear-associating DNA had a guanine plus cytosine (GC) content of 52%; unlike host DNA it was synthesized in the presence of mitomycin C, it could be removed from nuclei by centrifugation through sucrose, and it was susceptible to nuclease digestion. This nuclear-associating DNA appeared to be a precursor of cytoplasmic DNA of infected cells. The formation of the latter DNA class could be selectively inhibited by conditions (infection at 37 C or inhibition of protein synthesis) that permit continued incorporation of thymidine into nuclear-associating DNA. The cytoplasmic DNA class also had a GC content of 52%, was resistant to nuclease degradation, and its sedimentation profile in sucrose gradients corresponded to that of infective virus. Contrary to previous reports, we found that (i) viral DNA synthesis can continue in the absence of concomitant protein synthesis, and (ii) viral DNA synthesis is not abolished at 37 C. The temperature lesion in FV-3 replication appeared to be in the packaging of DNA into the form that appears in the cytoplasmic fraction of disrupted cells.     

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.     

Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Permissive Monkey Kidney Cells

Integration of simian virus 40 (SV40) deoxyribonucleic acid (DNA) into cellular DNA occurred when permissive African green monkey kidney (CV-1) cells were infected at a low multiplicity of SV40 in the presence of cytosine arabinoside.