The solubility characteristics of SV 40 tumor antigen

The solubility characteristics of SV40 tumor (T) antigen were investigated. Cell nuclei were isolated from cover glass cultures of SV 40-infected or transformed cells by detergent treatment. When treated in a buffered isotonic sucrose solution, the isolated nuclei contained typical SV40 T antigen by immunofluorescence. If the detergent was dissolved in an isotonic electrolyte solution containing 0.15 M monovalent cations (K+), the T antigen was extracted from the nuclei. Complement fixation tests demonstrated that the various solutions had no deleterious effects on the antigenicity of T antigen. Under identical conditions, the SV 40 capsid (V) antigen was not extracted from infected cell nuclei. These solubility characteristics place SV40 T antigen in the class of “loosely bound” nuclear proteins which includes non-histone proteins that appear to have cell regulatory functions. This supports the suggestion that the biological function of T antigen may be regulatory in nature.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest.

To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40 degrees, to complete ongoing replication and returned to 32 degrees, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40 degrees without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (alpha-32p)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI- restricted SV40 DNA, demonstrating partial replicon synchronization. The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.     

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.     

DNA synthesis in heterokaryons obtained by the fusion of polymorphonuclear leukocytes and cultured cells possessing different proliferative potentials

Heterokaryons between terminally differentiated polymorphonuclear leukocytes (PL) and culture cells of different proliferative potentials: mouse and rat embryo fibroblasts (EFM, EFR); immortal cells NIH 3T3 and E2; malignant cells NCC2, L929, He239 and SV 3T3,--were obtained by means of electrofusion. Radioautographic study of 3H-thymidine incorporation in the nuclei of heterokaryons showed that all the cells taken for fusion were able to induce reactivation of DNA synthesis in PL nuclei, however, with different rates: 7-37% for EFM and NIH 3T3 and 20-40% for malignant cells. The presence of oncogenes Elan in E2 cells and ras in NCC2 cells increased the rate of PL reactivation approximately twice as compared with the cells of original lines (EFR and NIH 3T3, correspondingly). In parallel to reactivation of DNA synthesis in PL nuclei inhibition of the synthesis in culture cell nuclei in the same heterokaryons was found. The rate of inhibition was about 70% for non-malignant and 23, 40 and 18% for NCC2, L and SV 3T3 cells, respectively. He239 cells, transformed by a temperature-dependent mutant of virus SV40 showed at permissive temperature the increased capacity of inducing reactivation of PL nuclei, though He239 cells susceptibility to inhibitory action of PL nuclei did not change with temperature. According to the behaviour in heterokaryons PL were found to be similar to chick erythrocytes, but differing from them by a pronounced inhibiting effect upon DNA synthesis in the nuclei of malignant cells.     

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin.

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.     

Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Maturation of replicating simian virus 40 DNA molecules in isolated nuclei by continued bidirectional replication to the normal termination region.

Mature SV40 DNA synthesized for different periods of time either in isolated nuclei or in intact cells was highly purified and then digested with restriction endonucleases in order to relate the time of synthesis of newly replicated viral DNA to its location in the genome. Replication in nuclei supplemented with a cytosol fraction from uninfected cells was a faithful continuation of the bidirectional process observed in intact cells, but did not exhibit significant initiation of new replicons. SV40 DNA replication in cells at 37 degrees C proceeded at about 145 nucleotides/min per replication fork. In the absence of cytosol, when DNA synthesis was limited and joining of Okazaki fragments was retarded, bidirectional SV40 DNA replication continued into the normal region where separation yeilded circular duplex DNA molecules containing one or more interruptions in the nascent DNA strands. In the presence of cytosol, this type of viral DNA was shown to be a precursor of covalently closed, superhelical SV40 DNA, the mature from of viral DNA.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

New host cell system for regulated simian virus 40 DNA replication.

Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.     

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.