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.     

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.     

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 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. 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 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.     

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.     

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 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.     

The adenovirus type 2-simian virus 40 hybrid virus Ad2+ND4 requires deletion variants to grow in monkey cells.

The Ad2+ND4 virus is an adenovirus type 2 (Ad2)-simian virus 40 (SV40) recombination. The Ad2 genome of this recombinant has a rearrangement within early region 3; Ad2 DNA sequences between map positions 81.3 and 85.5 have been deleted, and the SV40 DNA sequences between map positions 0.11 and 0.626 have been inserted into the deletion in an 81.3-0.626 orientation. Nonhybrid Ad2 is defective in monkey cells; however, the Ad2+ND4 virus can replicate in monkey cells due to the expression of the SV40-enhancing function encoded by the DNA insert. Stocks of the Ad2+ND4 hybrid were produced in primary monkey cells by using the progeny of a three-step plaque purification procedure and were considered to be homogeneous populations of Ad2+ND4 virions because they induced plaques in primary monkey cells by first-order kinetics. By studying the kinetics of plaque induction in continuous lines (BSC-1 and CV-1) of monkey cells, we have found that stocks (prepared with virions before and after plaque purification) of Ad2+ND4 are actually heterogeneous populations of Ad2+ND4 virions and Ad2+ND4 deletion variants that lack SV40 and frequently Ad2 DNA sequences at the left Ad2-SV40 junction. Due to the defectiveness of the Ad2+ND4 virus, the production of progeny in BSC-1 and CV-1 cells requires complementation between the Ad2+ND4 genome and the genome of an Ad2+ND4 deletion variant. Since the deletion variants that have been obtained from Ad2+ND4 stocks do not express the SV40-enhancing function in that they cannot produce progeny in monkey cells, we conclude that they are providing an Ad2 component that is essential for the production of Ad2+ND4 progeny. These data imply that the Ad2+ND4 virus is incapable of replicating in singly infected primary monkey cells without generating deletion variants that are missing various amounts of DNA around the left Ad2-SV40 junction in the hybrid genome. As the deletion variants that arise from the Ad2+ND4 virus are created by nonhomologous DNA recombination, the generation of deletion variants in monkey cells infected with Ad2+ND4 may be a useful model for studying this process.     

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.     

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.     

Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses.

The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2-infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ND2, Ad2+ND4, and Ad2+ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.     

Mapping of Simian Virus 40 Early Functions on the Viral Chromosome

The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2(+)ND(4), is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R(1) restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2(+)ND(4), called Ad2(+)ND(4del), was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R(1) endonuclease cleavage point.     

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.     

Density differences between hybrid and nonhybrid particles in two adenovirus-simian virus 40 hybrid populations

The adenovirus 7-simian virus 40 hybrid virus population E46⁺ was subjected to fixed-angle equilibrium density gradient centrifugation in CsCl. A difference in buoyant density between the hybrid virion and its nonhybrid adenovirus 7 counterpart was noted, the hybrid virion possessing the lower buoyant density. This difference in buoyant density appeared to be accentuated in a population of adenovirus 2^+t7, a derivative of E46⁺ in which the adenovirus 7-simian virus 40 genome had been transferred to an adenovirus 2 capsid.