Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells.

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.     

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. 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.     

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.     

Discrete regions of simian virus 40 large T antigen are required for nonspecific and viral origin-specific DNA binding.

The nondefective adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses, Ad2+ND2 and Ad2+ND4, have been used to determine which regions of the SV40 genome coding for the large tumor (T) antigen are involved in specific and nonspecific DNA binding. Ad2+ND2 encodes 45,000 M4 (45K) and 56,000 Mr (56K) T antigen-related polypeptides. The 45K polypeptide did not bind to DNA, but the 56K polypeptide bound nonspecifically to calf thymus DNA, Ad2+ND4 encodes 50,000 Mr (60K), 66,000 Mr (66K), 70,000 Mr (70K), 74,000 Mr (74K), and 90,000 Mr (90K) T antigen-related polypeptides, all of which bound nonspecifically to calf thymus DNA. However, in more stringent assays, where tight binding to viral origin sequences was tested, only the 90K protein specified by Ad2A+ND4 showed specific high affinity for sequences at the viral origin of replication. From these results and previously published experiments describing the SV40 DNA integrated into these hybrid viruses, it was concluded that SV40 early gene sequences located between 0.39 and 0.44 SV40 map units contribute to nonspecific DNA binding, whereas sequences located between 0.50 and 0.63 SV40 map units are necessary for specific binding to the viral origin of replication.     

Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells.

A comparison of the proteins synthesized in human cells at late times after infection with adenovirus (Ad2) and with the adeno-simian virus 40 (SV40) hybrid viruses revealed polypeptides of 30,000 and 92,000 molecular weight specific for the hybrid viruses Ad2+ND1 and Ad2+ND4, respectively. Cell-free translation of SV40-specific mRNA, prepared from these cells by hybridization of total cytoplasmic RNA to SV40 DNA, showed that the mRNA's specifying these two polypeptides were at least partially encoded by the SV40 portion of the hybrid viruses. Cell-free translation of SV40-specific mRNA prepared from monkey cells infected with SV40 produced polypeptides of 40,000, 43,000, 48,500, and 92,000 molecular weight. The SV40 and Ad2+ND4 92,000-molecular-weight polypeptides made in vitro were very similar in electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to the polypeptide precipitated by Tegtmeyer (1974) with SV40 anti-T serum.     

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 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.     

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 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.     

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 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.     

Similar regulation of the synthesis of adenovirus fiber and of simian virus 40-specific proteins encoded by the helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del.

Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40-specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses.     

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.