Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene.

Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.     

Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen.

Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed.     

trans-dominant defective mutants of simian virus 40 T antigen.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.     

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.     

Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40

We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8 degrees C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8 degrees C. The proliferation phenotypes of the dl-884-transformed cells at 39.8 degrees C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.     

A mutant SV40 large T antigen interferes with nuclear localization of a heterologous protein

A mutant SV40 genome carrying a frameshift at the carboxyl terminus of the large T antigen failed to replicate SV40 DNA and to transform rat2 cells, although the altered region is known to be dispensable for these functions. The mutant T antigen also failed to localize normally in the nucleus and interfered with nuclear localization of at least one other nuclear protein, adenovirus fiber. A double mutant carrying an additional lesion in the nuclear localization signal was also localized in the cytoplasm, but regained the ability to transform rat2 cells and no longer affected the nuclear localization of fiber protein. We suggest that the frameshift T antigen may disrupt a mechanism required for nuclear localization of proteins.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen.

Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.     

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.     

Abortive transformation of temperature-sensitive mutants of rat 3Y1 cells by simian virus 40: effect of cellular arrest states on entry into S phase and cellular proliferation

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, representing independent complementation groups, cease to proliferate predominantly with a 2n DNA content, at the restrictive temperature (39.8 degrees C) (temperature arrest) or at the permissive temperature (33.8 degrees C) at a confluent cell density (density arrest) (Ohno et al., 1984). We studied the temperature- or the density-arrested cells of these mutants infected with simian virus 40 (SV40) or its mutants affecting large T or small t antigen with respect to kinetics at 39.8 degrees C of entry into S phase and cellular proliferation. Three mutants, 3Y1tsD123, 3Y1tsF121 and 3Y1tsG125, expressed T antigen and entered S phase at 39.8 degrees C from both the arrested states after infection with either wild-type, tsA mutants, or a .54/.59 deletion mutant of SV40, whereas in the density-arrested 3Y1tsH203, expression of T antigen and entry into S phase were inefficient and ts. Following the WT-SV40 induced entry into S phase, the temperature-arrested 3Y1tsD123 detached from the substratum with no detectable increase in cell number, whereas the density-arrested ones completed a round of the cell cycle and then detached. 3Y1tsF121 and 3Y1tsG125 in the both arrested states proliferated through more than one generation. 3Y1tsF121 and 3Y1tsG125 in the density-arrested state infected with tsA mutants once proliferated and then ceased to increase in number as the percentage of T-antigen positive population decreased. These results suggest that wild-type and tsA-mutated large T antigens are able to overcome the cellular ts blocks of entry into S phase in the 3 ts mutants of 3Y1 cells in both the arrested states, and that small t antigen is not required to overcome the blocks. It is also suggested that cellular behaviors subsequent to S phase (viability, mitosis, and proliferation in the following generations) depend on cellular arrest states, on traits of cellular ts defects, and on the duration of large T antigen expression.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

Transformation of African Green Monkey Kidney Cells by Irradiated Adenovirus 7-Simian Virus 40 Hybrid

The ability of adenovirus 7-simian virus 40 (SV40) hybrid (strain LL "E-46") to replicate decreased exponentially as a function of the amount of gamma-irradiation; the ability to induce SV40 and adenovirus 7 T antigen decreased at a much slower rate. Nevertheless, the virus was still able to transform African green monkey kidney cells at a radiation dosage that had completely destroyed its replication ability. All transformed colonies were positive for SV40 T antigen but were negative for adenovirus 7 T antigen. The adenovirus 7-SV40 hybrid transformed cells were superinfectible with SV40 virus. Two of the three transformed cell populations apparently did not sensitize hamsters against the appearance SV40 primary tumors, thus suggesting a deficiency in the SV40 transplantation antigen.     

Simian virus 40 deletion mutants that transform with reduced efficiency.

We have constructed two simian virus 40 (SV40) early-region deletion mutants that lack a significant portion of the sequences normally used to encode the SV40 large tumor antigen. Despite these deletions, the mutants were able to transform mouse cells in a focus assay, although with a frequency that was drastically reduced relative to wild-type SV40. Cell lines expanded from the mutant-transformed foci contained integrated mutant DNA, expressed an SV40 tumor antigen (small-t), and exhibited a range of transformed phenotypes, which included the ability to grow while suspended in soft agar. We also present evidence that these mutants are defective for abortive transformation in an assay that tested the transient loss of anchorage dependence. Their ability to stably transform, contrasted with their inability to abortively transform at detectable levels, raises the possibility that the mechanism by which these mutants transform may be different from that of wild-type SV40.     

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.     

Evolutionary variants of simian virus 40 which are impaired in early lytic functions but transform nonpermissive cells

From an undiluted passaged virus stock, two size classes of defective simian virus 40 (SV40) DNA were isolated from which two evolutionary variants were cloned. By means of restriction enzyme and heteroduplex analysis, physical maps of the mutants have been constructed. Both mutants contained the region of SV40 DNA coding for the early proteins plus some adjacent sequences (the region from 0.120 to 0.685 map unit, clockwise, on the standard SV40 DNA map). Furthermore, each mutant contained, in the form of two inverted repeats, four times the sequences from the region 0.625 to 0.685 map unit, clockwise. Some biological properties of the mutant DNA were examined, and we found that the mutant DNA (i) has, as compared with SV40 DNA, an impaired ability to induce T antigen in permissive and nonpermissive cells; (ii) does not complement a thermosensitive A mutant of SV40; (iii) replicates very inefficiently without a helper; and (iv), as an apparent contradiction, transforms nonpermissive baby rat kidney cells as well as SV40 DNA. A hypothetical mechanism for the expression of the mutant DNA that might explain the observed biological properties is presented.     

Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.     

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.