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.     

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.     

Multiple insertions and tandem repeats of origin-minus simian virus 40 DNA in transformed rat and mouse cells.

Stable simian virus 40 (SV40) transformation requires integration and expression of the early region of the SV40 genome. We have examined the amount and state of integrated viral DNA of SV40-transformed NIH 3T3 mouse and F2408 rat fibroblast lines generated by transfection with either wild-type or origin-defective SV40 DNA. A functional SV40 replication origin was not required for multiple inserts and partial-repeat structures to form in NIH 3T3 mouse transformants. In contrast, partial repeats in F2408 rat transformants were rare when the SV40 replication origin was intact and not detected at all when it was defective.     

Antigenic structure of simian virus 40 large tumor antigen and association with cellular protein p53 on the surfaces of simian virus 40-infected and -transformed cells.

The antigenic structure of simian virus 40 (SV40) large tumor antigen (T-ag) in the plasma membranes of SV40-transformed mouse cells and SV40-infected monkey cells was characterized as a step toward defining possible biological function(s). Wild-type SV40, as well as a deletion mutant of SV40 (dl1263) which codes for a truncated T-ag with an altered carboxy terminus, was used to infect permissive cells. Members of a series of monoclonal antibodies directed against antigenic determinants on either the amino or the carboxy terminus of the T-ag polypeptide were able to precipitate surface T-ag (as well as nuclear T-ag) from both SV40-transformed and SV40-infected cells. Cellular protein p53 was coprecipitated with T-ag by all T-ag-reactive reagents from the surface and nucleus of SV40-transformed cells. In contrast, T-ag, but not T-ag-p53 complex, was recovered from the surface of SV40-infected cells. These results confirm that nuclear T-ag and surface T-ag are highly related molecules and that a complex of SV40 T-ag and p53 is present at the surface of SV40-transformed cells. Detectable levels of such a complex do not appear to be present on SV40-infected cells. Both the carboxy and amino termini of T-ag are exposed on the surfaces of SV40-transformed and -infected cells. The possible relevance of the presence of a T-ag-p53 complex on the surface of SV40-transformed cells and its absence from SV40-infected cells is considered.     

Transformation of isolated rat hepatocytes with simian virus 40

Rat hepatocytes were transformed by simian virus 40 (SV40). Hepatocytes from two different strains of rats and a temperature-sensitive mutant (SV40tsA 1609), as well as wild-type virus were used. In all cases, transformed cells arose from approximately 50% of the cultures containing hepatocytes on collagen gels or a collagen gel-nylon mesh substratum. Cells did not proliferate in mock-infected cultures. SV40-transformed hepatocytes were epithelial in morphology, retained large numbers of mitochondria, acquired an increased nucleus to cytoplasm ratio, and contained cytoplasmic vacuoles. Evidence that these cells were transformed by SV40 came from the findings that transformants were 100% positive for SV40 tumor antigen expression, and that SV40 was rescued when transformed hepatocytes were fused with monkey cells. All SV40-transformed cell lines tested formed clones in soft agarose. Several cell lines transformed by SV40tsA 1609 were temperature dependent for colony formation on plastic dishes. Transformants were diverse in the expression of characteristic liver gene functions. Of eight cell lines tested, one secreted 24% of total protein as albumin, which was comparable to albumin production by freshly plated hepatocytes; two other cell lines produced 4.2 and 5.7%, respectively. Tyrosine aminotransferase activity was present in five cell lines tested but was inducible by dexamethasone treatment in only two. We conclude from these studies that adult, nonproliferating rat hepatocytes are competent for virus transformation.     

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.     

The cellular secretory pathway is not utilized for biosynthesis, modification, or intracellular transport of the simian virus 40 large tumor antigen.

Unlike most proteins, which are localized within a single subcellular compartment in the eucaryotic cell, the simian virus 40 (SV40) large tumor antigen (T-ag) is associated with both the nucleus and the plasma membrane. Current knowledge of protein processing would predict a role for the secretory pathway in the biosynthesis and transport of at least a subpopulation of T-ag to account for certain of its chemical modifications and for its ability to reach the cell surface. We have examined this prediction by using in vitro translation and translocation experiments. Preliminary experiments established that translation of T-ag was detectable with as little as 0.1 microgram of the total cytoplasmic RNA from SV40-infected cells. Therefore, by using a 100-fold excess of this RNA, the sensitivity of the assays was above the limits necessary to detect the theoretical fraction of RNA equivalent to the subpopulation of plasma-membrane-associated T-ag (2 to 5% of total T-ag). In contrast to a control rotavirus glycoprotein, the electrophoretic mobility of T-ag was not changed by the addition of microsomal vesicles to the in vitro translation mixture. Furthermore, T-ag did not undergo translocation in the presence of microsomal vesicles, as evidenced by its sensitivity to trypsin treatment and its absence in the purified vesicles. Identical results were obtained with either cytoplasmic RNA from SV40-infected cells or SV40 early RNA transcribed in vitro from a recombinant plasmid containing the SP6 promoter. SV40 early mRNA in infected cells was detected in association with free, but not with membrane-bound, polyribosomes. Finally, monensin, an inhibitor of Golgi function, failed to specifically prevent either glycosylation or cell surface expression of T-ag, although it did depress overall protein synthesis in TC-7 cells. We conclude from these observations that the constituent organelles of the secretory pathway are not involved in the biosynthesis, modification, or intracellular transport of T-ag. The initial step in the pathway of T-ag biosynthesis appears to be translation on free cytoplasmic polyribosomes. With the exclusion of the secretory pathway, we suggest that T-ag glycosylation, palmitylation, and transport to the plasma membrane are accomplished by previously unrecognized cellular mechanisms.     

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.     

Cell-to-cell communication competence in simian virus 40-transfected rat ovarian cells is reduced following tumor selection

Summary A pSV3neo-transfected rat ovarian cell line (SV-GC) was developed from a primary granulosa culture (GC) to study gap junctional intercellular communication (GJIC) during Simian virus 40 (SV40) transformation. SV-GC expressed SV40 large T-antigen (T-ag), grew indefinitely in culture without luteinization, was anchorage independent, and formed tumors in nude mice. Ultrastructural analysis identified abundant gap junctional membrane and suggested that SV-GC was arrested at an early stage of differentiation. Functional GJIC, measured by a dye transfer technique (gap FRAP), was comparable to that observed in normal granulosa cells, suggesting that the expression of T-ag alone was insufficient to reduce GJIC. However, there was approximately a 50% loss in the rate of GJIC in the nude mouse SV-GC-tumor derived and G418 selected cell line (T-SV-GC). SV-GC→T-SV-GC also resulted in a transition from migration of cells as an epithelial sheet to the dissociation of individual fibroblastoid cells. Tumor cell detachment was also seen in migrating malignant human (A2780 and 547) and rat (DC3) ovarian cell lines. Co-culture combinations of normal (GC)→transformed (SV-GC) → tumor-derived (T-SV-GC) cells indicated that the rate of heterologous GJIC was characteristic of the least communicating partner. Taken together, these data suggested that SV-GC → T-SV-GC represented progression toward metastasis with concomitant reduction of GJIC and adhesiveness. These sequentially derived cell lines may be a useful in vitro model system for studies focusing on the mechanisms involved in the detachment of cells during the progression of ovarian cancer.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen.

pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.     

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.     

Sister chromatid exchange in FR3T3 rat fibroblasts transformed by Simian virus 40

The frequency of Sister Chromatid Exchange (SCE) was determined at low (33 degrees C) and high (40.5 degrees C) temperatures in cell lines derived from FR3T3 rat fibroblast cells after transformation either with Wild-Type Simian Virus 40 (SV40-WT), with an origin-defective SV40 (SV40-ori-), or with the early temperature-sensitive mutant tsA30. Of these cell lines, SV40-WT-, SV40-ori--, and one class of tsA30-transformants (A-type) express the transformed phenotype both at 33 and 40.5 degrees C. The other tsA30-transformants (N-type) revert to a normal phenotype at high temperature. As compared with normal FR3T3 cells, all transformants exhibited, at 33 degrees C, increased numbers of metaphases with high SCE rates. At 40.5 degrees C, all cell lines which expressed a transformed phenotype (SV40-WT, tsA30 type A, SV40-ori-) exhibited substantially increased SCE rates. That this increase was not related to a possible induction of viral replication by BrdU, was proven by Southern blot analysis and by SCE data on SV40-ori--transformed cells. By contrast, no such temperature-induced increase of SCE rates was observed in tsA30-transformants of type N.     

Polyoma virus middle t antigen: a tumor progression factor.

Polyoma virus (PyV) deletion mutant dl23 (affecting both large T and middle t but not small t antigens) was used to study transformation of 3T3 rat cells. This mutant generated stable transformants in the agar assay at a frequency similar to that of wild-type virus (WT). However, WT-induced transformants were detected 3 weeks after infection, whereas those induced by the mutant could not be detected until 6 to 8 weeks after infection. In this respect, dl23 PyV behaved like WT simian virus 40 (SV40). Cells transformed by WT SV40 or by dl23 PyV were similar in all their transformed properties. Those transformed by WT PyV were different from the others on the basis of morphology, cell adhesion to the substrate, release of protease activity, efficiency of doubling in agar, growth rate, and time required for tumor formation. Saturation density, the ability to grow in agar, the serum requirement for cloning, and the ability to grow on a cell monolayer were similar for all transformants. Middle t antigen enhanced membrane alterations and growth rate of the transformed cells, shortening the time required for tumor formation in rats.     

Simian virus 40 integration sites in the genome of virus-transformed mouse cells.

To gain information on the specificity of simian virus 40 (SV40) integration in the genome of transformed cells, mouse 3T3 cells were transformed by a temperature-sensitive (ts) SV40 mutant, using high multiplicity of infection (MOI). Transformed cells were superinfected with wild-type (wt) virus at high MOI. Clones were isolated and fused with permissive BSC-1 cells to promote virus rescue. All rescued viruses were of the ts type only. When the high-MOI transformants were infected with 3H-labeled wt SV40, the amount of radioactivity associated with their nuclear fraction was found to be similar to that of 3T3 cells. 3T3 cells were then transformed by ts SV40 at low MOI and superinfected by wt virus at high MOI. Upon fusion with BSC-1 cells, most clones produced both ts and wt virus. These results suggest that the number of stable SV40 integration sites in the 3T3 genome is limited, since they can be saturated by transformation at high MOI. When the MOI is low, the sites are not saturated and a subsequent infection can lead to integration.     

Development of 3T3-like lines from Balb/c mouse embryo cultures: Transformation susceptibility to SV40

Balb/c mouse embryo cultures that are carried under a rigid transfer schedule that minimizes cell-cell contact evolve into permanent lines that possess properties very similar to 3T3. From the same embryo cultures, a transfer schedule where cell-cell contact is extensive leads to lines (Balb/3T12) that form multiple cell layers and have saturation densities up to 25-fold higher than the Balb/3T3 lines. All the lines are hypotetraploid. Comparison of the Balb/3T3 lines with 3T3 reveals similar morphology, ability to grow at high dilution, low saturation density, and high susceptibility to transformation by SV40 virus. The Balb/3T12 lines, which are continually maintained at high cell density, show little improvement in cloning efficiency. Infection with SV40 produces transformants that can be selected by their ability to form colonies at low cell densities. Using the appropriate selective system for each line, it appears that SV40 T antigen induction and transformation in Balb/3T3 and Balb/3T12 are comparable.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.