Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.     

Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.     

Isolation and characterization of simian virus 40 early region deletion mutants.

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.     

Stabilization of the 53,000-dalton nonviral tumor antigen is not required for transformation by simian virus 40.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent about one-half of the coding region for large T antigen. We present evidence here that F8dl is able to transform mouse cells in a focus assay and that cell lines derived from these foci exhibit fully transformed phenotypes, have integrated mutant genomes, and express mutant-encoded proteins. This result implies that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for the maintenance of transformation. In addition, we have found that cells fully transformed by F8dl produce a 53,000-dalton nonviral tumor antigen (p53) that is as unstable as the p53 of untransformed cells. From this result we infer that transformation by simian virus 40 does not require the stabilization of p53.     

A simian virus 40 dl884/tsA58 double mutant is temperature sensitive for abortive transformation

We have constructed a dl884/tsA58 double mutant and a t+T- early-region deletion mutant and have used these mutants to study the roles of the simian virus 40 tumor antigens (T and t) in transformation. Our major conclusions are that (i) although the mutant tsA58 is not temperature sensitive for the maintenance of transformation, the dl884/tsA58 double mutant is; (ii) small t antigen can provide at least one, but not all, of the functions required for the maintenance of transformation; and (iii) at least two different functions are required for the maintenance of simian virus 40 transformation.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

Two classes of transformation-deficient, immortalization-positive simian virus 40 mutants constructed by making three-base insertions in the T antigen gene

We constructed two mutants of simian virus 40 (SV40) by introducing a three-base duplication at AvaII cutting sites within the large T antigen coding region, and we examined these mutants for their abilities to replicate in monkey GC7 cells, to transform rat cell line 3Y1 cells, and to transform and immortalize primary cells from newborn rats. Neither of the mutants could replicate in GC7 cells. One mutant with the duplication at 0.335 SV40 map units (m.u.) (inA942) could transform 3Y1 cells, but the other mutant with the duplication at 0.636 m.u. (inA941) could not. The two mutants could not transform primary rat cells but retained immortalization activity. The results suggest that transformation of primary cells by SV40 requires at least two distinct activities of the large T antigen, one of which can be replaced by a cellular function(s) expressed in immortalized 3Y1 cells.     

Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.     

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.     

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.     

Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).     

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.     

The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53.

The large T antigen encoded by simian virus 40 (SV40) plays essential roles in the infection of permissive cells, leading to production of progeny virions, and in the infection of nonpermissive cells, leading to malignant transformation. Primary mouse embryo fibroblasts (MEFs) are nonpermissive for SV40, and infection by wild-type SV40 leads to immortalization and transformation of a small percentage of infected cells. We examined the ability of an extensive set of mutants whose lesions affect SV40 large T antigen to immortalize MEFs. We found that immortalization activity was retained by all mutants whose lesions are located upstream of codon 346. This includes a mutant lacking amino acids 168 to 346. We previously showed (M. J. Tevethia, J. M. Pipas, T. Kierstead, and C. Cole, Virology 162:76-89, 1988) that sequences downstream of amino acid 626 are not required for immortalization of primary MEFs. Studies by Thompson et al. (D. L. Thompson, D. Kalderon, A. Smith, and M. Tevethia, Virology 178:15-34, 1990) indicate that all sequences upstream of residue 250, including the domain for binding of tumor suppressor protein Rb, are not required for transformation of MEFs. Together, these studies demonstrate that the immortalization activity of large T antigen for MEFs maps to sequences between 347 and 626. Several mutants with lesions between 347 and 626 retained the ability to immortalize at nearly the wild-type frequency, while others, with small insertions at amino acid 409 or 424 or a deletion of residues 587 to 589, failed to immortalize. The abilities of mutant T antigens to form a complex with tumor suppressor protein p53 were examined. We found that all mutants able to immortalize retained the ability to complex with p53, while all mutants which lost the ability to immortalize were no longer able to bind p53. This suggests that inactivation of the growth-suppressive properties of p53 is essential for immortalization of MEFs.     

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.     

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.