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.     

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.     

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.     

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.     

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.     

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.     

Location and identification of the genes for adenovirus type 2 early polypeptides

Virus-specific RNA was prepared from cells early after adenovirus type 2 infection and fractionated by hybridization to specific fragments of viral DNA. The viral mRNA was used to program cell-free protein synthesis, and the products were analyzed by electrophoresis. The genes for the early polypeptides of apparent molecular weight 44,000, 15,000, 72,000, 15,500, 19,000, and 11,000 daltons were located, respectively, between positions 0–4.1, 4.1–16.7, 58.5–70.7, 75.9–83.4, 89.7–98.6, and 89.7–98.6 of the conventional adenovirus DNA map. The polypeptide of molecular weight 72,000 daltons was shown to be the single-strand DNA-binding protein described by others. RNAs from three different adeno-transformed cell lines each program the synthesis in vitro of predominantly the 15K polypeptide, as well as variable amounts of the polypeptide of molecular weight 44,000 daltons. The genes for these two polypeptides are located in the portion of DNA known to be required for transformation of rodent cells by adenovirus.     

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.     

Integration and expression of viral DNA in cells transformed by host range mutants of adenovirus type 5.

Group I host range (hr) mutants of adenovirus type 5 are unable to transform rat embryo or rat embryo brain cells but induce an abnormal transformation of baby rat kidney cells. We established several transformed rat kidney cell lines and characterized them with respect to the transformed phenotype and the structure of the integrated viral DNA. The hr mutant-transformed cells, unlike wild-type virus transformants, were fibroblastic rather than epithelial, failed to grow in soft agar, and were also less tumorigenic in nude mice. Studies on the structure of the integrated viral DNA sequences showed that hr-transformed cells always contained the left end of the adenovirus DNA, but the size of the integrated DNA fragment varied among different lines, and a high percentage of the lines contained the entire viral genome colinearly integrated. The patterns of integration were maintained after prolonged growth in culture and after subcloning. Attempts to rescue infectious virus from lines which contained the entire genome were unsuccessful. Using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we analyzed the viral proteins expressed in hr-transformed cells. Results of these studies indicated that, like wild type-transformed cells, hr transformants expressed E1B proteins of molecular weight 58,000 and 19,000.     

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.     

Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.

The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.     

In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.     

Purification of a native membrane-associated adenovirus tumor antigen.

A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro.     

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.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Nuclear factor I is specifically targeted to discrete subnuclear sites in adenovirus type 2-infected cells.

During the S phase of the eukaryotic cell cycle and in virus-infected cells, DNA replication takes place at discrete sites in the nucleus, although it is not clear how the proteins involved in the replicative process are directed to these sites. Nuclear factor I is a cellular, sequence-specific DNA-binding protein utilized by adenovirus type 2 to facilitate the assembly of a nucleoprotein complex at the viral origin of DNA replication. Immunofluorescence experiments reveal that in uninfected cells, nuclear factor I is distributed evenly throughout the nucleus. However, after a cell is infected with adenovirus type 2, the distribution of nuclear factor I is dramatically altered, being colocalized with the viral DNA-binding protein in a limited number of subnuclear sites which bromodeoxyuridine pulse-labeling experiments have identified as sites of viral DNA replication. Experiments with adenovirus type 4, which does not require nuclear factor I for viral DNA replication, indicate that although the adenovirus type 4 DNA-binding protein is localized to discrete nuclear sites, this does not result in the redistribution of nuclear factor I. Localization of nuclear factor I to discrete subnuclear sites is therefore likely to represent a specific targeting event that reflects the requirement for nuclear factor I in adenovirus type 2 DNA replication.     

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.     

Viral gene products in adenovirus type-2 transformed hamster cells.

I have analyzed viral gene products expressed in five adenovirus type 2 (Ad2)- cytoplasmic, viral RNA which was selected by hybridization to cloned restriction endonuclease fragments of Ad2 DNA. Proteins synthesized in vitro were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and compared with those directed by RNAs prepared from productively infected cells. The early regions E1 and E4 of adenovirus type 2 (Ad2) were found to be expressed in all of five Ad2-transformed hamster embryo cells lines. RNA transcribed from early region E2, which codes for the 72,000-molecular-weight (72K) DNA-binding protein was detected in cell line HE1 only, and early region E3 was expressed exclusively in cell line HE4. RNA transcribed from the region between approximately 12 and 35 map units, coding for immediate early (13.5K, 52/53K) and immediate early proteins (13.6K, 16K, 17K, 87K), as well as RNA from late genes, was not found in any of the cell lines HE1 to HE5 had electrophoretic mobilities similar to those programmed by RNA from productively infected cells.