Relationship Between Virus-Induced Cellular Deoxyribonucleic Acid Synthesis and Transformation by Simian Virus 40

The fraction of cells in a confluent 3T3 cell monolayer induced by simian virus 40 infection to replicate deoxyribonucleic acid and divide corresponds to those cells which eventually become transformed. Virus-induced cells were partially separated from noninduced cells by sedimentation through Ficoll gradients. Three- to eightfold higher transformation frequencies were obtained with those cells that began to synthesize cellular deoxyribonucleic acid and divide shortly after simian virus 40 infection as compared to noninduced cells.     

Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.     

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.     

Inducible expression of amplified human beta interferon genes in CHO cells.

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene. Levels of expression of 1 U/cell per day were achieved on superinduction, giving corresponding titers of up to 10(10) U/liter medium in culture supernatants. Constitutive production of IFN-beta rates of about 0.5% of superinduced rates was observed; cells producing these levels of IFN-beta had acquired resistance to cytotoxic antiviral effects of IFN-beta. Two forms of human IFN-beta were produced; a major glycosylated 23,000-dalton form and an unglycosylated 18,500-dalton form. The latter had greatly reduced antiviral activity. IFN-beta production was very sensitive to cellular growth rate; the highest levels were produced by density-arrested cultures. Regulation of IFN-beta production by polyriboinosinic acid-polyribocytidylic acid or by cell density effects required the presence of DNA sequences 5' to the IFN-beta-coding sequences; replacement of these sequences with the simian virus 40 early promoter resulted in uninducible, density-independent production of IFN-beta.     

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid.

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.     

Transformation of Mouse Macrophages by Simian Virus 40

Studies were undertaken to prove that simian virus 40 (SV40) can transform the mouse macrophage, a cell type naturally restricted from deoxyribonucleic acid (DNA) replication. Balb/C macrophages infected with SV40 demonstrated T-antigen production and induced DNA synthesis simultaneously. In the absence of apparent division, these cells remained T antigen-positive for at least 45 days. SV40 could be rescued from nondividing, unaltered macrophages during the T antigen-producing period. Proliferating transformants appeared at an average of 66 days post-SV40 infection. Established cell lines were T antigen-positive and were negative for infectious virus, but yielded SV40 after fusion with African green monkey kidney cells. Their identity as transformed macrophages was substantiated by evaluation of cellular morphology, phagocytosis, acid phosphatase, beta(1c) synthesis, and aminoacridine incorporation.     

Shuttle vectors conferring hygromycin B resistance toE. coli and to mammalian cells

Shuttle vectors expressing resistance to hygromycin B in bothE. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of theneo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive inE. coli.Abbreviations HM hygromycin - hpt hygromycin phosphotransferase gene - neo neomycin (geneticin) phosphotransferase gene - DHFR dihydrofolate reductase     

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.     

Polynucleotide Ligase Activity in Cells Infected with Simian Virus 40, Polyoma Virus, or Vaccinia Virus

The conversion of simian virus 40 (SV40) component II deoxyribonucleic acid to component I has been used to assay polynucleotide ligase in extracts of tissue culture cells. All cell types examined, including chicken, hamster, mouse, monkey, and human cells, contained adenosine triphosphate-dependent ligase. After infection of mouse embryo, monkey kidney, and HeLa cells with polyoma virus, SV40, and vaccinia virus, respectively, the enzyme activity increased, but its cofactor requirement was unchanged. In vaccinia virus-infected cells, the increased activity was localized in the cytoplasm. Ligase induction occurred in the presence of cytosine arabinoside but was prevented by puromycin. Rifampicin blocked the production of infectious vaccinia particles but had little effect on the induction of ligase.     

Isolation of Simian Virus 40 Recombinants from Cells Infected with Oligomeric Forms of Simian Virus 40 Deoxyribonucleic Acid

Oligomeric forms of simian virus 40 (SV40) deoxyribonucleic acid (DNA) were isolated from monkey kidney cells infected with two plaque morphology mutants of SV40. Recombinant, large clear-plaque-type SV40 was produced in cells productively infected with oligomeric forms of SV40 DNA.     

Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Permissive Monkey Kidney Cells

Integration of simian virus 40 (SV40) deoxyribonucleic acid (DNA) into cellular DNA occurred when permissive African green monkey kidney (CV-1) cells were infected at a low multiplicity of SV40 in the presence of cytosine arabinoside.     

Generation of helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene.

We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.     

Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.     

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.