Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

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.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VII. Characterization of the Simian Virus 40 RNA Species Induced by Five Nondefective Hybrid Viruses

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.     

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.     

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.     

Integrated Simian Virus 40 DNA: Nucleotide Sequences at Cell-Virus Recombinant Junctions

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.     

Primate's p53 inhibits SV40 DNA replication in vitro

Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication in vitro as well as in vivo while that from primate cells does not (1-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication in vitro. p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication in vitro. These results suggest that inhibition of SV40 DNA replication by p53 should be regarded as general property of the protein and does not determine the mode of species specific replication of SV40 DNA.     

Cloning and sequencing of viral integration site in human fibroblasts immortalized by simian virus 40.

We have analyzed cellular DNA sequences at the viral genome integration site in a human fibroblast cell line VA13 immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNA sequences did not show any homology to the DNA sequences previously reported. This suggests that immortalization by SV40 was not induced by the destruction of any known oncogene or anti-oncogene at the integration site. We did not find the precise substantial sequence homology at the junctional site between the cellular DNA and SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNA abutting SV40 DNA was found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNA is now under study.     

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.     

Replication of SV40 chromatin in extracts from eggs of Xenopus laevis.

Simian virus 40 (SV40) nucleoprotein complexes were prepared from lytically infected cells and used as primer-templates for DNA replication in protein extracts from Xenopus eggs. We found that nucleoprotein containing replicating SV40 DNA served as primer-template while nucleoprotein with nonreplicating SV40 DNA was ineffective. In vitro DNA synthesis begins with short DNA fragments ("Okazaki fragments") which are, in later steps, joined to give unit length SV40 DNA strands, suggesting that in vivo initiated rounds of replication are completed in vitro in the Xenopus system. This conclusion is supported by a restriction enzyme analysis showing that in vitro DNA synthesis occurs in fragments distal to the SV40 origin of replication. Our studies indicate that SV40 DNA replication in Xenopus extracts can be used an an experimental system to study the biochemistry of replicative DNA chain elongation in vitro.     

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.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IV. Characterization of the Simian Virus 40 Ribonucleic Acid Species Induced by Wild-Type Simian Virus 40 and by the Nondefective Hybrid Virus, Ad2+ND1

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.