In Vitro Transformation by the Adenovirus-Simian Virus 40 Hybrid Viruses: V. Virus-Specific Ribonucleic Acid in Cell Lines Transformed by the Adenovirus 2-Simian Virus 40 and Adenovirus 12-Simian Virus 40 Transcapsidant Hybrid Viruses

The ribonucleic acid-deoxyribonucleic acid hybridization technique was utilized to determine the presence of adenovirus (ad) and SV40 genetic information and to determine which ad genomes were present in clones of hamster cells transformed with the ad 2-SV40 and ad 12-SV40 transcapsidant hybrid virus populations. The results were correlated with the morphology of the transformed cells and colonies. It was found that cells transformed by either transcapsidant virus which had an SV40 morphology contained the ad 7 and SV40 genomes, whereas cells with a typical ad morphology contained only ad genetic information. Cells and colonies with morphological features of both ad- and SV40-transformed cells contained either the ad 2, or ad 12 genomes, depending on the transcapsidant used, together with the ad 7 and SV40 genomes. The results indicate the following: at least three different events occurred during transformation of hamster cells by the transcapsidant virus populations; the morphology of the resulting clones is determined by the viral genome(s) present; the linkage of the ad 7-SV40 genomes is confirmed since the ad 7- SV40 genomes were never found to be dissociated; the defective ad 7-SV40 genomes are capable of causing transformation; and the transcapsidant particle is probably composed of only ad 7 and SV40 genetic information.     

Induction of Cellular DNA Synthesis by the Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses

Induction of cellular DNA synthesis by the nondefective adenovirus 2-simian virus 40 hybrid viruses was examined in monkey and hamster cells. It was not possible to discriminate the effects of the hybrid viruses from those of wild-type adenovirus 2.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses. VI. Characterization of the DNA from Five Nondefective Hybrid Viruses

Certain biophysical characteristics of the DNA from each of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND(1), Ad2(+)ND(2), Ad2(+)ND(3), Ad2(+)ND(4), Ad2(+)ND(5)) have been determined. The guanine plus cytosine content varied from 55 to 57% and was not significantly different from that of nonhybrid Ad2 (56%), and the hybrid DNA molecules had mean molecular lengths which were similar to that of the standard, Ad2. The Ad2 and SV40 components of each hybrid were linked by alkali-resistant, presumably covalent bonds. The percentage of SV40 DNA in each hybrid virus was determined by hybridization with SV40 complementary RNA in a calibrated system. The results indicate that each hybrid virus DNA contains a different percentage of SV40 nucleotide sequences. The estimated size of the SV40 DNA component varies from 48,000 daltons for Ad2(+)ND(3) to 840,000 daltons for Ad2(+)ND(4), the latter being equivalent to between one-fourth and one-third of the SV40 genome.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VII. Characterization of the Simian Virus 40 RNA Species Induced by Five Nondefective Hybrid Viruses

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IX. Template Topography in the Early Region of Simian Virus 40

The DNAs of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses contain overlapping segments of the early region of wild-type SV40 DNA. The complementary DNA strands of these five viruses have been separated with synthetic polyribonucleotides in isopycnic cesium chloride gradients. The relative amounts of early and late SV40 template in the DNA of each virus were determined by RNA-DNA hybridization with late lytic SV40 RNA, which contains sequences complementary to both templates. From the distribution of early and late templates in the five overlapping SV40 segments, we conclude that either the entire early region of SV40 is symmetrically transcribed in vivo, or, more probably, that the early SV40 templates are not contiguous.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses IV. Characterization of the Simian Virus 40 Ribonucleic Acid Species Induced by Wild-Type Simian Virus 40 and by the Nondefective Hybrid Virus, Ad2+ND1

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses V. Isolation of Additional Hybrids Which Differ in Their Simian Virus 40-Specific Biological Properties

Four new nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated. Although these viruses (designated Ad2(+)ND(2), Ad2(+)ND(3), Ad2(+)ND(4), and Ad2(+)ND(5)) were clonal derivatives of the same Ad2-SV40 hybrid population, they differ significantly from each other and from the previously isolated nondefective hybrid, Ad2(+)ND(1), in their biological properties or in the amount of SV40-specific RNA induced during lytic infection.Like Ad2(+)ND(1), Ad2(+)ND(2), and Ad2(+)ND(4) pass serially in both human embryonic kidney (HEK) and primary African green monkey kidney cells. In contrast, Ad2(+)ND(3) and Ad2(+)ND(5) pass serially only in HEK cells. Ad2(+)ND(2) is like Ad2(+)ND(1) in that it induces the SV40 U antigen, but not SV40 T antigen; however, in contrast to the perinuclear SV40 antigen induced by Ad2(+)ND(1), the SV40 antigen induced by Ad2(+)ND(2) is located peripherally in the cytoplasm as well as in the perinuclear region of infected cells. Ad2(+)ND(4) induces both the SV40 T and U antigens. Ad2(+)ND(3) and Ad2(+)ND(5) do not induce serologically detectable SV40 antigens and are distinguished from each other on the basis of the relative quantities of SV40-specific RNA which they induce. The induction of different SV40-specific functions suggests the incorporation of different segments of SV40 DNA within the genomes of the respective hybrid viruses.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Transformation of African Green Monkey Kidney Cells by Irradiated Adenovirus 7-Simian Virus 40 Hybrid

The ability of adenovirus 7-simian virus 40 (SV40) hybrid (strain LL "E-46") to replicate decreased exponentially as a function of the amount of gamma-irradiation; the ability to induce SV40 and adenovirus 7 T antigen decreased at a much slower rate. Nevertheless, the virus was still able to transform African green monkey kidney cells at a radiation dosage that had completely destroyed its replication ability. All transformed colonies were positive for SV40 T antigen but were negative for adenovirus 7 T antigen. The adenovirus 7-SV40 hybrid transformed cells were superinfectible with SV40 virus. Two of the three transformed cell populations apparently did not sensitize hamsters against the appearance SV40 primary tumors, thus suggesting a deficiency in the SV40 transplantation antigen.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses II. Relationship of Adenovirus 2 Deoxyribonucleic Acid and Simian Virus 40 Deoxyribonucleic Acid in the Ad2+ND1 Genome

A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2(+)ND(1) virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2(+)ND(1) molecule. From the results obtained, it was estimated that 1% of the Ad2(+)ND(1) DNA consists of SV40 nucleotide sequences.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VIII. Association of Simian Virus 40 Transplantation Antigen with a Specific Region of the Early Viral Genome

Two of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrids induce SV40 transplantation resistance in immunized hamsters. These two hybrids, Ad2(+)ND(2) and Ad2(+)ND(4), contain 32 and 43% of the SV40 genome, respectively. The pattern of induction of SV40 transplantation antigen (TSTA) by the various hybrids differentiates TSTA from both SV40 U and T antigens. Since the SV40 RNA induced by both these hybrids is early SV40 RNA, these findings confirm that TSTA is an early SV40 function. By combining available data on SV40 antigen induction by these hybrids with electron microscopy heteroduplex mapping studies, the DNA segment responsible for the induction of SV40 TSTA can be inferred to lie in the region between 0.17 and 0.43 SV40 units from the site on the SV40 chromosome cleaved by E. coli R(1) restriction endonuclease.     

Studies on Nondefective Adenovirus-Simian Virus 40 Hybrid Viruses I. A Newly Characterized Simian Virus 40 Antigen Induced by the Ad2+ND1 Virus

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen-the SV40 "U" antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T(+)U(+) sera) and those that react with SV40 T antigen only (T(+)U(-) sera). SV40 U-specific sera from monkeys immunized with Ad2(+)ND(1)-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T(+)U(+) hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2(+)ND(1) virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.     

Adenovirus Type 2-Simian Virus 40 Hybrid Population: Evidence for a Hybrid Deoxyribonucleic Acid Molecule and the Absence of Adenovirus-Encapsidated Circular Simian Virus 40 Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) from the adenovirus-encapsidated particles of the adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid population plaque variant (Ad2(++) HEY), known to yield SV40 virus with high efficiency, was studied by equilibrium density centrifugation followed by ribonucleic acid-DNA hybridization employing virus-specific complementary ribonucleic acids synthesized in vitro. These techniques establish linkage between the Ad2 and SV40 components in the adenovirus-encapsidated particles of this population. The linkage is alkali-resistant and presumably covalent; thus, the Ad2 DNA and SV40 DNA are present in a hybrid molecule. Velocity centrifugation studies in alkaline sucrose gradients eliminated the possibility that supercoiled circular SV40 DNA is present in the adenovirus capsids. The DNA obtained from the adenovirus-encapsidated particles of the Ad2(++) HEY population appears to consist of nonhybrid Ad2 DNA and Ad2-SV40 hybrid DNA molecules.     

Analysis of Simian Virus 40-induced Transformation of Hamster Kidney Tissue In Vitro: V. Variability of Virus Recovery from Cell Clones Inducible with Mitomycin C and Cell Fusion

Clones of simian virus 40-transformed hamster kidney cells from which infectious virus could be recovered and clones which did not yield virus by the overlay technique were subjected to chemical (mitomycin C) induction and to co-cultivation and fusion studies with indicator cells. The results of these studies indicate that the complete viral genome is present in all clones tested but that considerable heterogeneity with respect to inducibility exists among the clones. It is suggested that differences either in the number of viral genomes per transformed cell or in the status of the viral genome in transformed cells exist among the various clones. Furthermore, the inducibility of the clones may be correlated with their malignant potential.     

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.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses III. Base Composition, Molecular Weight, and Conformation of the Ad2+ND1 Genome

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), differs from the defective Ad-SV40 hybrid populations previously described, in that this hybrid virus can replicate without the aid of nonhybrid adenovirus helper. Consequently, the deoxyribonucleic acid (DNA) from this virus, which can be obtained free of nonhybrid adenovirus DNA, is well suited for biophysical studies on Ad-SV40 hybrid DNA. Such studies have been performed and demonstrate Ad2(+)ND(1) DNA to have a buoyant density (1.715 g/cm(3)) and thermal denaturation profile (T(m) = 75.1 C) almost identical with nonhybrid Ad2 DNA. Furthermore, its molecular weight, as determined by analytical zone sedimentation and electron microscopy, was 22 x 10(6) to 25 x 10(6) daltons, which is also very similar to that determined for Ad2. Electron micrographs showed all of the hybrid molecules to be double-stranded and linear. By using this determination of the molecular weight of Ad2(+)ND(1) DNA and assuming that 1% of this molecule consists of covalently linked SV40 DNA (see companion paper), we calculate that the hybrid DNA molecule contains 220 x 10(3) to 250 x 10(3) daltons of SV40 DNA, or the equivalent of one-tenth of the SV40 genome.     

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear.