State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.     

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ~1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.     

Integrated polyoma genomes in inducible permissive transformed cells.

Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells.     

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.     

Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant.

The polyoma virus hr-t deletion mutant A185, when compared to wild-type (Py) virus, is at least 10⁵ fold inhibited in its transforming ability. Total cellular DNA from 50 cell lines derived from individual colonies formed after infection of Rat-1 cells with A185 virus was analyzed for the presence of viral sequences by “blot” hybridization (Southern, 1975). Viral sequences were detected in two of these cellular DNAs. One positive cell line (18–37) was studied in detail. The viral sequences present in 18–37 cells as well as the viral sequences present in virus rescued from 18–37 after fusion with permissive mouse cells were identified as A185 and not Py sequences. The A185 viral sequences in 18–37 cells were found to exist both covalently linked to host DNA sequences (integrated) and as free forms. The integrated A185 viral sequences were present in a partial head-to-tail tandem array, as has been observed for Py sequences in transformed rat cells (Birg et al., 1979). Both integrated and free forms of A185 viral sequences were retained in subclones of the parental 18–37 cell line although a simplification of the integrated viral sequence was observed. In the 18–37 cells the 100K large T antigen was synthesized but the 55K middle and 22K small T antigen species were not detected. The 18–37 cells had a normal morphology, were density-sensitive, anchorage-dependent and did not form tumors when injected into syngeneic animals. This normal phenotype of the 18–37 cells was not a result of the inability of the cells to express the transformed phenotype, since the 18–37 cells could be transformed at a high frequency upon infection with Py virus. These results show that integration of viral sequences per se or the presence of the 100K large T antigen is not sufficient for the transformed phenotype to be expressed, and strongly suggest that Py-induced transformation is mediated by the 55K middle and/or 22K small T antigens.     

Stability of polyoma DNA sequences and virus-coded proteins during tumor formation

To determine the stability of polyoma viral DNA in transformed rat cells during their growth in vivo, we compared the state and arrangement of polyoma virus DNA sequences in virus-transformed rat cell lines before and after their passage in vivo. In cell lines from 12 independent tumors induced by the inoculation of animals with three different transformed cell lines, we could detect no significant changes in the arrangement of viral DNA sequences associated with the in vivo passage of these cell lines. In 13 of 14 tumor cell lines examined, the pattern of polyoma virus tumor antigens, characterized by the presence of the polyoma virus large, middle, and small tumor antigens, was unchanged.     

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.     

Loss of functional large T-antigen and free viral genomes from cells transformed in vitro by polyoma virus after passage in vivo as tumor cells.

We have analyzed the state, arrangement, and expression of polyoma viral DNA sequences in a number of in vitro-transformed Fischer rat cells before and after growth in vivo as tumour cells. When the in vitro lines used to induce the tumors contained only a single insert of viral sequences and did not produce either a full-size 100,000-dalton (100K) large T-antigen or free viral genomes, no differences in the above-mentioned properties were observed. By contrast, in vitro cell lines containing multiple inserts of viral sequences, a functional 100K large T-antigen, and free viral genome induced tumor cells which displayed a reduced number of inserts of viral sequences and which did not produce either a functional 100K large T-antigen or free viral genomes. All of the in vitro lines and their tumor cell derivatives expressed the polyoma virus 55K middle and 22K small T-antigen species. Possible mechanisms for the selection in vivo against cells containing a functional 100K large T-antigen and consequently free viral genomes are discussed.     

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.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.     

Evidence for integrated EBV genomes in Raji cellular DNA.

Human lymphoid cell lines cannot be grown in long-term tissue culture, as a rule, unless the cells have been transformed by Epstein-Barr virus (EBV). The latent EBV DNA in established cell lines, is mainly present as free covalently closed circles but viral DNA sequences with properties of integrated DNA also seem to be present. We have extended the studies on the physical state of the EB viral DNA sequences in the cell line Raji which appear at a lower density than that for free EB viral DNA during fractionation on CsCl density gradients. In such material a novel EcoRI EBV DNA fragment is present, which hybridizes to viral sequences homologous to EcoRI A. This fragment is not present in free covalently closed circular EBV DNA. When this EcoRI fragment is further analysed with HindIII a smaller fragment than expected, which contains BamHI W sequences, is detected. The demonstration of this HindIII fragment and its characteristics as a joint, viral-host chromosome fragment will be discussed.