Equilibrium Density Gradient Studies on Simian Virus 40-Yielding Variants of the Adenovirus Type 2-Simian Virus 40 Hybrid Population

The simian virus 40 (SV40)-yielding variants of the adenovirus type 2 (Ad.2)-SV40 hybrid (Ad.2(++)) population were studied by means of fixed-angle equilibrium density gradient centrifugation in cesium chloride. The hybrid virions of the Ad.2(++) high-efficiency yielder population banded at densities of 0.004 g/cm(3) lighter than the nonhybrid Ad.2 virions. The degree of separation of the hybrid particles was sufficient to permit greater than 100-fold relative purification by two cycles of centrifugation. Hybrid particles that produce adenovirus plaques in African green monkey kidney cells by two-hit kinetics (one-hit kinetics when assayed on lawns of nonhybrid adenovirus) were not separable from the particles that yield SV40 virus. The hybrid particle in the Ad.2(++) low-efficiency yielder population was not separable from the nonhybrid Ad.2 virions.     

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 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.     

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.     

Two adenovirus type 2-simian virus 40 hybrid populations that contain the entire SV40 genome differ in their ability to induce SV40 transplantation immunity in hamsters but not in mice.

Ad2++ HEY and Ad2++ LEY are two adenovirus 2(Ad2)-simian virus 40 (SV40) hybrids distinguished by differences in the efficiency with which they produce SV40 progeny in lytically infected African green monkey kidney cells. These virus populations are composed of nonhybrid Ad2 and hybrid virions, the majority of which contain more than 1 unit of SV40 DNA. The Ad2++ HEY and LEY populations also differ in their ability to induce SV40 transplantation immunity in rodents. Only Ad2++ HEY induces SV40 transplantation immunity in hamsters, whereas both viruses induce significant SV40 transplantation immunity in adult BALB/c mice.     

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. 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.     

Simian virus 40 DNA replication, transcription, and antigen induction during infection with two adenovirus 2-SV40 hybrids that contain the entire SV40 genome.

The Ad2++hey hybrid virus population produces simian virus 40 (SV40) efficiently during lytic infection, whereas Ad2++ley does not, although both hybrids contain a complete SV40 genome. In this report, we demonstrate the synthesis of nonhydrid SV40 DNA in Ad2++HEY-infected Vero cells, but only early SV40 RNA is transcribed efficiently in Ad2++LEY-infected cells. Ad2++HEY induces SV40 U, T, and V antigens during lytic infection of African green monkey kidney cells, whereas Ad2++LEY induces only SV40 U and T antigens. These variations in the behavior of Ad2++HEY and Ad2++LEY regarding expression of SV40 functions probably reflect differences in the rate of SV40 excision from the hybrid genomes.     

Sequence of the junction in adenovirus 2-SV40 hybrids: examples of illegitimate recombination.

The nucleotide sequences of six Ad2-SV40 junctions from three Ad2-SV40 hybrid viruses (Ad2++HEY, Ad2++LEY and Ad2+D1) were determined. Comparison of parental adenovirus 2 and SV40 DNA sequences with the sequence at the Ad2-SV40 junctions revealed that 5 out of 6 junctions are abrupt transitions from Ad2 to SV40 DNA, and in one case (Ad2++LEY, right junction) there is an additional nucleotide at the junction, which cannot be ascribed to either DNA. Ad2++HEY and Ad2+D1 right junctions are identical and Ad2++LEY and Ad2+ND4 left junctions are identical, a result that strongly suggests these Ad2-SV40 hybrids arose by recombination between the linear Ad2 DNA and circular SV40 DNA, followed by recombination between Ad2 DNA and SV40 DNA present in the Ad2-SV40 hybrid DNA. The unambiguous transition of Ad2 DNA into SV40 DNA at the junction sites is an example of recombination events which have apparently occurred without any homology at the recombination site.     

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.     

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.     

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.     

Structure of two adenovirus-simian virus 40 hybrids which contain the entire SV40 genome

Two defective adenovirus-simian virus 40 hybrids which contain the entire SV40 genome (Ad2⁺⁺HEY and Ad2⁺⁺LEY)² have been isolated. Upon infection of cells permissive for SV40 both hybrids give rise to infectious SV40 virions, but with markedly different efficiencies. In the case of Ad2⁺⁺HEY nearly all cells infected with a hybrid particle yield SV40 progeny, whereas in the case of Ad2⁺⁺LEY infectious SV40 is produced in only about one in 10⁴ cells infected with hybrid particles. The structures of the DNA molecules in the Ad2⁺⁺HEY and Ad2⁺⁺LEY populations were examined using electron microscope heteroduplex methods. Both populations were found to be heterogeneous. Ad2⁺⁺HEY contained three hybrids (HEY-I, HEY-II, and HEY-III) whose genomes differed only in their content of SV40 DNA (0.45 ± 0.02, 1.43 ± 0.04, and 2.39 ± 0.09 SV40 genomes, respectively). Ad2⁺⁺LEY contained two hybrids (LEY-I and LEY-II), which also differed only in their content of SV40 DNA (0.03 ± 0.01 and 1.05 ± 0.01 SV40 genomes, respectively). In those hybrids which contained more than one complete SV40 genome (HEY-II, HEY-III, LEY-II) the excess SV40 DNA was shown to be organized as a tandem repetition. These data suggest that the various hybrid genomes within each population are interconvertible by recombination events, which insert or excise an SV40 genome. It is proposed that HEY-II and HEY-III yield infectious SV40 with higher efficiency than LEY-II because their SV40 DNA segments contain longer tandem repetitions; thus, the probability of an intramolecular recombination event which results in excision of an SV40 genome is greater.     

Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid

Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection.     

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.     

Use of Nondefective Adenovirus-Simian Virus 40 Hybrids for Mapping the Simian Virus 40 Genome

A series of viable recombinants between adenovirus 2 (Ad2) and simian virus 40 (SV40) (nondefective Ad2-SV40 hybrids) have been isolated. The members of this series (designated Ad2(+)ND(1) through Ad2(+)ND(5)) differ from one another in the early SV40-specific antigens and the SV40-specific RNA species which they induce in infected cells. They also contain different amounts of SV40 DNA as shown by RNA-DNA hybridization techniques. We have examined the structure of the DNA molecules from these hybrids, using electron microscope heteroduplex mapping techniques. Each hybrid was found to contain a single segment of SV40 DNA of characteristic size covalently inserted at a unique location in the adenovirus 2 DNA molecule. The SV40 segments of the various hybrids formed an overlapping series with a common end point. When the results of the electron microscopic study were combined with data on antigen induction, it was found that a self-consistent map could be constructed which related specific regions of the SV40 genome to the induction of specific antigens. The order of these early SV40 antigen inducing regions in the SV40 DNA segments contained in the nondefective hybrids is: U antigen, tumor specific transplantation antigen, and T antigen with the U antigen region being nearest the common end point.     

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.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.