Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

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.     

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.     

Administration of ethylnitrosourea to neonate hamsters increases growth and frequency of SV40-induced fibrosarcomas

The in vivo interaction between the chemical carcinogen ethylnitrosourea (ENU) and the oncogenic simian virus 40 (SV40) was studied. Inbred newborn Syrian golden hamsters were injected subcutaneously with SV40 (5 x 10(6) plaque-forming units), ENU (0.5% solution, 125 or 25 mg/kg body wt), or equal mixtures of the two. Animals that received SV40 and ENU developed more tumors (100% vs 52%) within a shorter latent period (10 weeks vs 18 weeks) than animals that received SV40 alone. Animals given SV40 and ENU showed increased mortality and increased metastatic tumors (54.2% vs 30.8%) compared with those given SV40 alone. The SV40 and ENU group also exhibited multiple (greater than 10 nodules) pulmonary metastases (33.3% vs 7.7%) and metastases in multiple organs (12.5% vs 0%) compared with animals injected with SV40 alone. No difference in primary tumor size, histology, and SV40 T-antigen content was detected between SV40- and SV40/ENU-induced tumors. Four weeks after SV40 or SV40 plus ENU treatment, animals were challenged intradermally with 2.7 x 10(6) SV40-transformed hamster cells. Five weeks after challenge, 89.5% of the animals treated with SV40 and ENU and 45.4% of animals treated with SV40 developed tumors at the challenge site. Newborn animals given SV40 and ENU developed larger tumors at the challenge site (P less than 0.002) than newborns treated with SV40 alone. Thus, administration of ENU to hamsters during the neonatal stage of development produced a long-lasting systemic effect that enhanced tumor development by transplanted SV40-transformed hamster cells.     

Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.     

Reinitiation Within One Cell Cycle of the Deoxyribonucleic Acid Synthesis Induced by Simian Virus 40

The infection of secondary cultures of Chinese hamster cells with simian virus 40 (SV40) induces the appearance of cells with polyploid deoxyribonucleic acid (DNA) content or chromosomal component within one cell generation. The mechanism of this phenomenon was studied by the use of 5-bromodeoxyuridine (BUdR) incorporation as a DNA density marker. When cultures were treated with (14)C-BUdR and colcemide and harvested at 48 hr postinfection, only hybrid and light DNA molecules were found in control cultures, whereas in infected cultures there were also heavy molecules. The proportion of heavy DNA synthesized during the experimental period varied from 13 to 25%. It was determined by DNA-DNA hybridization that the heavy DNA consisted of cellular DNA. In radioautographic experiments, it was shown that, under the conditions used, a fraction of the infected cell population twice replicated its complete DNA content. Analysis of the kinetics indicated that the heavy DNA resulted from the reinitiation of DNA synthesis after the initial replication of the entire cell DNA. It was concluded that, after infection with SV40, a fraction of the Chinese hamster cell population undergoes two cycles of DNA synthesis without intervening mitosis.     

Virus-Specific Deoxyribonucleic Acid in Simian Virus 40-Exposed Hamster Cells: Correlation with S and T Antigens

Several homologous hamster embryonic cell lines, transformed in association with simian virus (SV) 40 infection, were examined for the presence of deoxyribonucleic acid (DNA) complementary to SV40 ribonucleic acid (RNA) made in vitro. The methods employed permitted the detection of 10(-5) mug of viral DNA in 100 mug of cellular DNA, corresponding to one-fifth of an SV40 DNA molecule per cell. Those lines which contained both the SV40 surface (S) and tumor (T) antigens also contained DNA complementary to SV40 RNA synthesized in vitro. In contrast, neither of two lines which contained S, but not T, antigen contained detectable DNA complementary to SV40 RNA. These findings suggest that the production of S antigen does not depend upon the persistence of SV40 DNA in transformed cells.     

Analysis of Simian Virus 40-Induced Transformation of Hamster Kidney Tissue in Vitro VIII. Induction of Infectious Simian Virus 40 from Virogenic Transformed Hamster Cells by Amino Acid Deprivation or Cycloheximide Treatment

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.     

Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts.

A threonine phospholipid in polyoma virus-transformed hamster embryo fibroblasts has been characterized as phosphatidylthreonine. The identification has been made by chemical and enzymatic hydrolysis. Acid hydrolysis of the phospholipid produces free threonine. Mild alcoholysis produces a water-soluble derivative having the properties of glycerophosphorylthreonine. Hydrolysis with phospholipase C produces phosphorylthreonine which on prolonged acid hydrolysis yields threonine. Phosphatidylthreonine in the cell is more accessible to reaction with fluorodinitrobenzene than is phosphatidylserine. Phosphatidylthreonine also has been found as a major aminophospholipid in two other polyoma-transformed hamster cell lines and in the BHK-21/C13 line including the PVT-3 and TS-3 lines. the latter derived from BHK cells. Only a trace amount of phosphatidylthreonine occurs in normal liver, kidney and spleen of the adult mouse, in normal liver and kidney of the adult hamster, in whole mouse and hamster embryos, and in mouse 3T3 cells and SV40-transformed 3T3 cells.     

Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells

We studied the pattern of integration of the simian virus 40 (SV40) genome into the cellular DNA of N-transformants (temperature sensitive) and A-transformants (temperature insensitive) derived from 3T3-Fisher rat and Chinese hamster lung cells. The SV40 DNA was covalently linked to the cellular DNA in both types of transformants. In the rat cells, most N-transformants contained SV40 sequences integrated at a single site; most A-transformants contained SV40 sequences integrated at two to five sites. In the Chinese hamster cells, no significant correlation between the number of integration sites and the phenotype of the transformant was found; one of three integration sites were observed for both the N- and A-transformants. Single copies and tandem repeats of SV40 sequences were observed in A- and N-transformants derived from rat cells. A-transformants arise neither by amplification of the SV40 genome nor by integration at a unique site.     

Transformation of bone marrow cells in rodents by recombinant plasmid pBRSV

Bone marrow cells (mouse strain CBA/Ca and Syrian hamster cells) were transformed with pBRSV DNA containing T-antigen of the SV40 virus. The SV40 T-antigen in transformed cell was detected in 0.5% cases by immunofluorescence with specific antibodies. Extrachromosomal localization of recombinant DNA was shown by means of retransformation of E. coli cells with cytoplasmic spleen DNA from mice previously injected intravenously the transformed bone marrow cells.     

Herpes simplex virus infection generates large tandemly reiterated simian virus 40 DNA molecules in a transformed hamster cell line.

When the simian virus 40 (SV40)-transformed Syrian hamster cell line Elona is infected with herpes simplex virus type 1, an excessive amplification of SV40-specific DNA sequences occurs. Analysis of total DNA from herpes simplex virus-infected cells revealed that amplified DNA sequences were present predominantly in a high-molecular-weight form, consisting of a tandem array of many unit-length SV40 DNA molecules. Repeat units of amplified DNA were found to be very similar to standard SV40 DNA as was shown by restriction analyses, except for a small deletion close to the origin of replication, which could also be detected in the chromosomal DNA of uninfected cells. A procedure, devised for selective enrichment of amplified SV40 DNA molecules from the bulk of cellular and herpesviral DNA, allowed molecular cloning of single repeat units and nucleotide sequence analysis of the relative genomic region.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate stimulates simian virus 40 induction by DNA-damaging agents and tumor initiators.

Simian virus 40 (SV40)-transformed Syrian hamster kidney cells produce infectious SV40 virus particles after treatments which damage DNA, such as UV irradiation or mitomycin C treatment. We have found that the induction of SV40 by DNA-damaging agents is greatly stimulated when a typical tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), is present in the medium. Phorbol, which has a molecular structure similar to TPA but does not have any tumor-promoting activity, showed no such stimulatory effect on SV40 induction. This apparent synergistic effect of DNA-damaging agents and tumor promoter (TPA) was more pronounced when a tumor initiator, benzo [a]pyrene or 2-acetamido-fluorene, was combined with TPA. The effect of TPA on UV-triggered SV40 induction was greatly influenced by the timing of TPA addition to the culture medium, which was most efficient when addition of TPA was 5 to 20 h before UV irradiation. The effect of TPA, however, was not observed in SV40 rescue from hamster cells by cell fusion with permissive monkey (C7) cells.     

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.     

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.