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.     

Regulation of expression and chromosomal subunit conformation of avian retrovirus genomes.

We have investigated the copy number, chromosomal subunit conformation and regulation of expression of integrated avian retrovirus genomes. Our results indicate that there are approximately two copies of the endogenous viral genomes (RAV-O) per haploid cell genome in uninfected chick embryo fibroblasts (CEF) and red blood cells (RBC). The copy number and subunit conformation (as measured by DNAasel sensitivity) of the RAV-O genomes are independent of the level of expression of these viral DNA sequences. In cells isolated from embryos of the V+, gs-chf- and gs+chf+ phenotypes, approximately one of the two viral genomes is in a DNAase l-sensitive conformation. Upon infection with an exogenous Rous sarcoma virus (PR-RSV-C), one new viral genome is integrated per haploid CEF genome. The newly integrated RSV genome is completely sensitive to DNAase l, and the subunit conformation of the endogenous viral genomes is not altered by the integration of additional exogenous proviruses. Both the endogenous and newly integrated exogenous viral genomes are present in "nu-body" structures, and the selective sensitivity of these proviral DNA sequences to DNAase l is maintained in isolated nucleosomes. Our experiments revealing the DNAase l sensitivity of one of the two RAV-O genomes in gs-chf-CEF led us to reexamine the level of viral specific RNA in CEF of various GS genotypes. We find that GS/GS CEF contain approximately 100 copies of viral RNA per cell, gs/gs CEF contain no detectable viral RNA, and the heterozygote GS/gs CEF contain approximately 50 copies of viral specific RNA per cell. These results suggest that the GS gene controls production of RAV-O RNA sequences in CEF in a "cis" fashion. In RBCs, however, the expression of the RAV-O genome is independent of the GS gene, with both GS/GS and gs/gs RBCs containing roughly equivalent amounts of viral specific RNA. Our results suggest that the chromosomal structure of the endogenous viral genes is independent of the GS gene, and that the GS gene is cis-acting and tissue-specific.     

Production and characteristics of seven rat embryo cell lines transformed by adenovirus type 5 and its DNA

Seven cell lines transformed by adenovirus type 5 and its DNA were obtained. It was shown that different cell lines contain the fragments of viral DNA which differ in length and number of copies per DNA of diploid cells. They contain from the left end 6% of the viral DNA to complete or almost complete viral genome. All studied cell lines were sensitive to reinfection with adenovirus type 5. They produced no virus being cocultivated with cell sensitive to the virus. No cell line was able to induce tumors even in immunosuppressed newborn rats. All cell lines formed colonies in soft agar. The level of virus-specific antigens was higher in cells that contained a large part of the viral genome. The methods used did not allow to correlate the biological properties of the transformed cells with the length and the number of copies of the integrated part of the viral genome.     

Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.

The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2: separated strands of fragments of ³²P-labelled adenovirus 2 DNA, generated by cleavage with restriction endonucleases EcoR1, Hpa1 and BamH1, were added to reaction mixtures at sufficient concentrations to drive hybridizations with infected or transformed cell RNA. Under these conditions, the fraction of ³²P-labelled DNA entering hybrid is directly proportional to the absolute amount of complementary RNA in the reaction.At 8 hours after infection in the presence of cytosine arabinoside, “early” viral messenger RNA sequences are present at a frequency of 300 to 1000 copies per cell. The abundance of early mRNA sequences in different lines of adenovirus 2-transformed rat cells is markedly lower than their concentration in lytically infected cells. Moreover, the abundance of early mRNA in a given transformed rat cell line reflects the number of copies of its template DNA sequences per diploid quantity of cell DNA. After the onset of the late phase of the lytic cycle, the abundance of one early mRNA species, that coding for a single-stranded DNA binding protein required for viral DNA replication, is amplified. Viral RNA sequences complementary to regions of the genome coding for other early mRNA sequences remain at the level observed at 8 hours after infection.Exclusively “late” viral mRNA sequences are present over a range of concentrations, 500 to 10,000 copies per cell, depending on the region of the genome. By 18 hours after infection, the nucleus contains approximately three times as much total, viral RNA as the cytoplasm. The abundant nuclear, viral RNA sequences at 18 hours are transcribed from a contiguous region, 65% of the genome in length. In some cases, viral RNA sequences complementary to mRNA sequences are very abundant in the nucleus. When cytoplasmic and nuclear fractions are mixed and incubated under annealing conditions, some mRNA sequences will anneal with more abundant, anti-messenger nuclear RNA sequences to form double-stranded RNA. Such annealing of nuclear, viral RNA to early, cytoplasmic mRNA sequences probably accounts for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.     

Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells.

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.     

Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2.

Complementary strand-specific adenovirus DNA of full length or from endonuclease BamHI fragments was used as a probe to estimate the fractional representation and abundance of viral sequences in five hamster cell lines (Ad2HE1-5) transformed with UV-inactivated adenovirus type 2. The fraction of the viral genome present in the five transformed cell lines varied from 44% in the Ad2HE5 cell line to 84% in the Ad2HE3 cell line. The number of viral DNA copies per diploid cell equivalent ranged from 1.8 in the Ad2HE1 line to 7.1 in the Ad2HE4 line. In vivo labeling with [35S]methionine followed by immunoprecipitation with an antiserum against adenovirus type 2 early proteins revealed virus-specific polypeptides with molecular weights of 42,000 to 58,000 in extracts from all five hamster cell lines. Several other early viral polypeptides were detected in some of the adenovirus type 2-transformed hamster cell lines.     

Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.     

Viral DNA in transformed cells: II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease from Escherichia coli strain RY-13 (Yoshimori, 1972) (EcoRI) or restricting endonuclease from Hemophilus parainfluenzae (Hpa I) and the resulting fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from nine lines of adenovirus 2-transformed rat cells and from control cells. Six of the transformed cell lines contained viral DNA sequences homologous to two of the seven Hpa I⁴ fragments and to part of one of the six EcoRI fragments. From the order of the fragments formed by EcoRI and Hpa I on the adenovirus 2 map we conclude that these cell lines contain only the segment of viral DNA that stretches from the left-hand end to a point about 14% along the viral genome. Thus, any viral function expressed in transformed cells must be coded by this small section of viral DNA. The three remaining lines of adenovirus 2-transformed rat cells are more complicated and contain not only the sequences from the left-hand end of the viral DNA, but also other segments of the viral genome. However, no adenovirus 2-transformed rat cell contained DNA sequences homologous to the complete viral genome.     

Transformation with specific fragments of adenovirus DNAs. II. Analysis of the viral DNA sequences present in cells transformed with a 7% fragment of adenovirus 5 DNA

Five clones of rat kidney cells transformed by a small restriction endonuclease fragment of adenovirus 5 (Ad5) DNA (fragment HsuI G, which represents the left terminal 7% of the adenovirus genome) were analyzed with respect to the viral DNA sequences present in the cellular DNAs. In these analyses, the kinetics of renaturation of 32P-labeled specific fragments of Ad5 DNA was measured in the presence of a large amount of DNA extracted either from each of the transformed cell lines or from untransformed cells. The fragments were produced by digestion of 32P-labeled adenovirus 5 DNA with endo R.HsuI, or by digestion of 32P-labeled fragment HsuI G of adeno 5 DNA with endo R.HpaI. All five transformed lines were found to contain DNA sequences homologous to 75--80% of Ad5 fragment HsuI G only. Clones II and V contained approximately 48 copies per quantity of diploid cell DNA, clone VI about 35 copies, clone IV 22 copies and clone III 5--10 copies. These results indicate that a viral DNA segment as small as 5.5% of the Ad5 genome, contains sufficient information for the maintenance of transformation.     

Two "early" mRNA species in adenovirus type 2-transformed rat cells

mRNA isolated from adenovirus 2-infected HeLa cells at early times during the productive cycle and from two lines of adenovirus 2-transformed rat embryo cells (F17 and T2C4) was fractionated on sucrose gradients after disaggregation. Viral mRNA species were identified by hybridization across such gradients with the separated strands of restriction endonuclease fragments of 32P-labeled DNA known to be complementary to adeovirus 2 "early" and adenovirus 2-transformed cell mRNA. mRNA transcribed from the left-hand 14% of the adenovirus 2 genome was found to comprise two species, 16 to 17S and 20 to 21S: the same sized mRNA's were present both at early times during productive infection and in the two transformed rat cell lines. Direct comparison of the sequences present in these two mRNA species by additional saturation hybridizations suggests that they are not related to one another. Three additional regions of the adenovirus 2 genome, all of which are located in the right-hand 40% of the adenovirus 2 genome, are complementary to early mRNA sequences: each of these appears to specify one major mRNA species of about 22S. Thus, five major species of adenovirus type 2 early mRNA have been identified. Two of these, copied from the left-hand 14% of the viral genome, are also present in adenovirus 2-transformed rat cells.     

Sequence organization of a viral DNA insertion present in the adenovirus-type-5-transfonned hamster line BHK268-C31

The hamster cell line BHK268-C31 contains two large viral inserts which both include sequences from the left-hand end of adenovirus type 5 (Ad5) DNA. One of these viral inserts has been cloned in the λ vector Charon 4A. Electron microscopic analysis and restriction enzyme mapping shows that the recombinant carries a 4.4-kb-long colinear segment of viral DNA, which is located between map positions 1.5 and 14.2 in the Ad5 genome. The junctions between viral DNA and flanking sequences have been sequenced and found not to show any specific features. One of the junctions is located in the E1 a coding region, 573 bp from the left-hand end of the Ad5 genome, whereas the other junction is situated in the coding region for polypeptide IVa2. The promoter region as well as the cap site for the mRNAs from region E la are thus missing from this insert and its role in viral transformation is unclear.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Transformation of primary rat kidney cells by DNA fragments of weakly oncogenic adenoviruses.

Primary cultures of baby rat kidney (BRK) cells were transformed by intact DNA and DNA fragments of weakly oncogenic human adenovirus types 3 and 7. The smallest fragment found to contain transforming activity was the left-terminal 4% endo R.HindIII fragment (for both adenovirus type 3 and 7 DNAs). The efficiency of transformation of this fragment was low, and no permanent cell line could be established. Left-terminal fragments ranging from 84 to 4,5% of the viral genome could all transform BRK cells with the same efficiency as intact viral DNA. A number of adenovirus type 7 DNA fragment-transformed lines were established and were found to contain persistent viral DNA sequences and adenovirus subgroup B-specific T antigen. Consequently, the transforming functions of adenovirus types 3 and 7 are located at the extreme left-hand end of the genome, and the minimum size for a DNA fragment with transforming activity is 1.0 X 10(6) daltons. These results do not rule out the possibility that viral genes located outside the transforming region may also influence transformation.     

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Comparison of viral RNA sequences in adenovirus 2-transformed and lytically infected cells

The complementary strands of fragments of ³²P-labelled adenovirus 2 DNA generated by cleavage with restriction endonucleases EcoRI or Hpa1 were separated by electrophoresis. Saturation hybridization reactions were performed between these fragment strands and unlabelled RNA extracted from the cytoplasm of adenovirus 2-transformed rat embryo cells or from human cells early after adenovirus 2 infection. The fraction of each fragment strand complementary to RNA from these sources was measured by chromatography on hydroxylapatite. Maps of the viral DNA sequences complementary to messenger RNA in different lines of transformed cells and early during lytic infection of human cells were constructed.Five lines of adenovirus 2-transformed cells were examined. All contained the same RNA sequences, complementary to about 10% of the light strand of EcoRI fragment A. DNA sequences coding for this RNA were more precisely located using Hpa1 fragments E and C and mapped at the left-hand end of the genome. Thus any viral function expressed in all adenovirus 2-transformed cells, tumour antigen, for example, must be coded by this region of the viral genome. Two lines, F17 and F18, express only these sequences; two others, 8617 and REM, also contain mRNA complementary to about 7% of the heavy strand of the right-hand end of adenovirus 2 DNA; a fifth line, T2C4, contains these and many additional viral RNA sequences in its cytoplasm.The viral RNA sequences found in all lines of transformed cells are also present in the cytoplasm of human cells during the early phase of a lytic adenovirus infection. The additional cytoplasmic sequences in the 8617 and REM cell lines also correspond to “early” RNA sequences.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.