Structure of integrated simian virus 40 DNA in transformed mouse cells

The structure of integrated viral DNA sequences in four lines of simian virus 40 (SV40)-transformed Balb/c 3T3 cells has been probed using restriction endonucleases and the Southern (1975) transfer method. By considering data from a large number of restriction digests of DNA from each line, and by using a novel method of handling the data, we have constructed fairly detailed physical maps of the integrated DNA in each line. The most striking of the features of the maps described here is that none is easily explained by the integration of a single SV40 genome into the DNA of the host cell. Three of the lines contain at least two distinct integrated segments and the fourth contains a single segment longer than the viral DNA. Considered individually, only two of the seven segments that we have mapped might be unit length. Of the remaining five, two are longer and three are shorter than the viral genome. It seems likely, therefore, that at least in SV40-transformed Balb/c 3T3 cells simple, single integrations are rare.The endpoints of these seven segments of integrated DNA fall at many positions distributed over the entire genome, confirming earlier studies (Ketner &; Kelly, 1976; Botchan et al., 1976), which indicated that SV40 integration is not absolutely site-specific.Finally, one of the lines mapped here (SVB209) does not possess an intact SV40 early region, an observation that suggests the possibility that a normal viral large T polypeptide is not synthesized by this line.     

Tandem integration of complete and defective SV40 genomes in mouse-human somatic cell hybrids.

We have analyzed the arrangement of SV40 DNA sequences integrated in human chromosome 7 in two lines of mouse-human somatic cell hybrids: one containing only one human chromosome 7 per cell and the other containing an average of about three. We found that the integration site differs in both the viral and host sequences in the two clones. However, the sites of integration into the several copies of human chromosome 7 of one clone are identical. Each chromosome 7 in both clones carries approximately six viral genomes tandemly linked. Some of these genomes lack about 20% of the DNA from the late region, including the Eco RI site.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Quantitation of the viral DNA present in somatic cell hybrids between mouse and SV40-transformed human cells

Recent studies of somatic cell hybrids between mouse cells and SV40-transformed human cells have demonstrated a correlation between the expression of SV40 T-antigen and the presence of human chromosome 7. We have used two types of nucleic acid hybridization procedures to detect and quantitate the presence of viral DNA sequences in the DNA of the hybrid cell clones. Results of reassociation kinetics as well as hybridization with a single-strand probe indicate that SV40 DNA is present only in those hybrid clones which both contain human chromosome 7 and express the SV40 T-antigen. SV40 DNA was not detectable either in the clones which had lost human chromosome 7, or in the rare clones which retain human chromosome 7 but which do not express T-antigen. We have thus extended the correlation between human chromosome 7 and the SV40 T-antigen to the presence of integrated SV40 DNA in somatic cell hybrid clones.     

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.     

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.     

Karyotype evolution of the human HBL-100 cell line and mapping of the integration site of SV40 DNA

Cytogenetic analysis of the human HBL-100 cell line, that we have previously shown to harbour SV40 genetic information (Caron de Fromentel et al., 1985), reveals numerous chromosomal rearrangements as soon as the 30th in vitro passage. The karyotype is relatively stable during in vitro maintenance and even at late passages (approximately 70) when the cells have acquired the capacity to form tumors in nude mice. In all the somatic cell hybrids obtained after fusion of mouse 3T3-4E cells with HBL-100 cells, several human chromosomes are maintained and a derivative from chromosome 15-der(15)- is the most frequently observed. The der(15) marker is present in the HBL-100 cell line at every passage studied as well as in different cell lines derived from tumors induced by HBL-100 cells. The various hybrids, originally isolated for a transformed phenotype on the basis of their ability to grow in soft-agar, were all found to express the SV40 T-antigen. In situ hybridization of an SV40 DNA probe to chromosome spreads obtained from one of these hybrids shows that the integration site of the viral genome is located on the der(15) marker chromosome, at band 15q24. The possible cooperation of SV40 T-antigen with some other oncogene(s), required by human HBL-100 cells in order to express a malignant phenotype, is discussed.     

Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C(0)t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36 degrees C) or nonpermissive (39.5 degrees C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration.     

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.     

Isolation of Two Plaque Variants from the Adenovirus Type 2-Simian Virus 40 Hybrid Population Which Differ in Their Efficiency in Yielding Simian Virus 40

Studies on the adenovirus type 2-simian virus 40 (SV40) hybrid population demonstrated two genetically stable variants within this population, which were isolated by plaquing in African green monkey kidney cells. These variants were similar in that each induced SV40 T antigen in human embryonic kidney cells and contained similar concentrations of nonhybrid adenovirus type 2 virions and adenovirus-encapsidated particles containing the infectious SV40 genome. These variants differed markedly, however, in their ability to produce SV40 viral antigen in human embryonic kidney cells and the efficiency with which they produce SV40 plaques in monkey cell monolayers. It is postulated that the differences in SV40-yielding efficiency between these variants lie in the nature of the recombinant deoxyribonucleic acid composing the genome of the hybrid particles.     

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.     

The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture

Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.     

Analysis of stable and unstable viral forms in SV40-infected human keratinocytes

We analyzed the state of the genomic DNA of the papovavirus SV40 in human keratinocytes as viral-infected cells gradually acquired a transformed phenotype over time. Initially, the vast majority of the viral DNA is maintained either in a full-length supercoiled form or as truncated subgenomic fragments with little evidence of integration. However, analyses of clonal populations revealed great heterogeneity and instability of the viral DNA, and we were able to isolate one clonal subpopulation in which integrated forms of the virus appeared to predominate. Similarly, uncloned populations eventually ceased production of the "free" viral DNA after several years in culture and instead came to display tandemly repeated SV40 copies at a single host integration site. Interestingly, Bg1 II digestion of host DNA generated restriction fragments containing the integrated SV40 DNA, which were of differing sizes in cultures at the 144th vs the 163rd serial passage suggesting modification or rearrangement of sequences at or near the integration site. Host sequences flanking the integrated viral DNA at the 163rd serial passage have been isolated on restriction fragments generated by Eco RI, Bam HI, and Hpa II digestion. These analyses suggest that the integrated virus is linearized near the Bg1 I site and contains a large deletion in the SV40 early region at one of the viral-host junctions.