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.     

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.     

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.     

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.     

Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12.

Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.     

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

Viral DNA in transformed cells: I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease isolated from Escherichia coli strain RY-13 (Yoshimori, 1971) and the resulting six fragments were separated by electrophoresis through polyacrylamide-agarose gels. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from a line of transformed rat cells and from control cells. The entire sequences of two of the fragments and part of the sequence of a third fragment were not detectable in the transformed cell DNA. Thus the line of adenovirus 2-transformed rat cells contains sequences homologous to only about 46% of the viral DNA. From the order of the fragments, formed by this restricting endonuclease on the adenovirus 2 map, it seems that the viral sequences that are absent from transformed cells form one continuous segment located in the center of the viral genome.     

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.     

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.     

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.     

Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns.

We investigated whether, upon the integration of multiple copies of adenovirus type 12 (Ad12) DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change with consecutive passages of cells in culture. By the injection of purified Ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, were established. These cell lines carried different copy numbers of Ad12 DNA per cell in an integrated form and differed in morphology. Cell line HT5 had been passed twice through hamsters as tumor cells and was subsequently passaged in culture. Patterns of Ad12 DNA integration were determined by restriction cleavage of the nuclear DNA with BamHI, EcoRI, HindIII, MspI, or PstI followed by Southern blot hybridization using 32P-labeled Ad12 DNA or its cloned terminal DNA fragments as hybridization probes. In this way, the off-size fragments, which represented the sites of linkage between Ad12 and cellular DNAs, were determined. At early passage levels in culture, the integration sites of Ad12 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the five different tumor cell lines. Upon repeated passage, however, the off-size fragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions, Ad12-transformed tumor cell lines that carry the foreign (Ad12) genome in selective, probably very similar sites of the cellular genome evolve.     

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.     

Nucleotide sequence analysis of the linked left and right hand terminal regions of adenovirus type 5 DNA present in the transformed rat cell line 5RK20.

A peculiar phenomenon is observed in several adenovirus type 2 or 5 (Ad2 or Ad5) transformed cell lines: the right hand and left hand terminal regions of the viral genome present in the viral DNA insertions of these cell lines are found to be linked together. A large part of the viral DNA insertion present in the Ad5 transformed rat cell line 5RK20 has been cloned in the lambda vector Charon21A, including the segment containing the linked terminal regions. Sequence analysis of the linkage region showed a perfect homology with the Ad5 DNA sequence and a direct linkage of basepair (bp) 63 of the left hand end of the viral genome to bp 108 of the right hand end. No cellular or rearranged viral sequences were present. Our findings suggest that the joining of viral sequences into the cellular genome.     

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.     

Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells

The patterns of integration of viral DNA in five lines of adenovirus type 2-transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet light-inactivated Ad2². In all lines, segments in the central parts of the viral genome are missing. The lines HE1, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively.The patterns of integration in lines HE2 and HE3 are identical; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral genomes. It is also conceivable that line HE2 may have undergone losses of integrated Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have deletions in parts of the EcoRI F and D fragments. The remainders of these fragments are linked to cellular DNA. The termini of the segments of the viral genome have been inverted and linked to each other. This linkage could have occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA.In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes.