Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus.

A large number of polyoma virus-transformed cells of rat, mouse, and hamster origin were examined for presence of T-antigen species. The results showed that all lines of cells contained middle and small T antigens, but not all contained a full-sized large T antigen, in some cell lines large T antigen was absent, whereas in others it was present as truncated forms lacking various lengths of the carboxy-terminal part of the protein. Cells transformed by the new viable deletion mutants of polyoma virus, dl-8 and dl-23, formed larger and smaller colonies or foci, respectively, when they were suspended in semisolid medium or plated as monolayers together with untransformed cells on a plastic surface. The deletions in the DNA of these mutants resulted in the shortening of the large and middle T antigens simultaneously without affecting the size of the small T antigen. Variation of large T-related proteins in dl-8 and dl-23-transformed cells seemed to be the same as that observed in wild-type-transformed cells. Regardless of the amount and size of large T-related protein in mutant-transformed cells, the phenotype of the cells was entirely dependent on the mutant used. The results suggest that (i) persistence of large T antigen is not universally required for the maintenance of the transformation phenotype, (ii) small T antigen alone may not be sufficient for inducing the full expression of the transformation phenotype, and (iii) middle T antigen is implicated as being primarily responsible for the full expression of the phenotype of transformation. The results also provide the evidence that the carboxy-terminal region of middle T antigen and a part of large T antigen are encoded in the genome in the same DNA segment around map units 88 to 94 in different reading frames.     

Immunization against the polyoma virus-induced tumor-specific transplantation antigen by early region mutants of the virus

To investigate the relation between the polyoma tumor-specific transplantation antigen and the virus-coded proteins, mice were immunized by inoculation of a variety of viable polyoma virus mutants and then challenged with polyoma virus-induced tumors. Two classes of early region mutants were used. One class produces a normal small T-antigen and truncated middle and large T-antigens. The second class (hr-t mutants) forms a normal large T-antigen together with N-terminal fragments of small and middle T-antigens. All mutants, transforming as well as nontransforming, induced protection against polyoma virus tumors. However, there were quantitive differences between the mutants. The finding that an hr-t mutant could induce tumor rejection suggests that full-length middle and small T-antigens are not necessary for the induction of this response. Since intact middle T-antigen is the only virus-coded protein known to associate with the plasma membrane, the possibility must be considered that the polyoma virus tumor-specific transplantation antigen consists of cellular components.     

A 61,000-dalton truncated large T-antigen is uniformly expressed in hamster cells transformed by polyomavirus

Various polyomavirus-transformed hamster cell lines derived from tumors or from infected hamster cell cultures synthesized polyoma middle and small tumor (T)-antigens but no full-size large T-antigen. Instead, all cell lines produced the same or similar polyoma T-antigen-related proteins of ca. 61 kilodaltons (kDal). Like large T-antigen synthesized in lytically infected mouse cells, the 61-kDal proteins were phosphoproteins showing electrophoretic and charge heterogeneities. Chromatographic analysis of the methionine-containing tryptic peptides indicated that the 61-kDal proteins were truncated forms of large T-antigen comprising amino acid residues 1 to 485 (+/- 25). Analysis of viral DNA present in hamster chromosomal DNA of three independently isolated cell lines confirmed that synthesis of the 61-kDal proteins was due to a discontinuity in the large T-antigen coding sequence, most likely located between 7 and 8.9 map units on the polyoma DNA map. The three cell lines yielded essentially the same patterns of viral DNA-containing restriction enzyme fragments, suggesting that insertion of viral DNA into the hamster chromosomes took place at closely similar sites.     

Polyoma T (tumor) antigen species in abortively and stably transformed cells

Stable neoplastic transformation of cells by polyoma virus requires the participation of two viral genes, designated ts-a and hr-t. The effects of mutations in these two genes on the patterns of T-antigen synthesis during productive infection have been previously described: ts- a mutants are affected in the “large” (100K) nuclear T antigen, and hr-t mutants are affected in the “middle” (36K, 56K, 63K) and “small” (22K) T agtigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, rrespectively. Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr-t gene.     

Role of the Three Polyoma Virus Early Proteins in Tumorigenesis

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.     

Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.

Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Characterization of different tumor antigens present in cells transformed by simian virus 40.

In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.     

Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.     

Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion.

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.     

Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.     

Transformation of hamster embryo cells by chymotrypsin-treated and untreated polyoma virus: characterization of transformants

The ability of chymotrypsin-treated (chymo+) and untreated (chymo-) polyoma virus to transform cultured hamster embryo fibroblasts was examined. The data show that exposure to this protease reduces the ability of the virus to transform non-permissive cells to essentially the same extent as it reduces its ability to replicate in permissive cells. Twenty-five lines of transformed cells were established from colonies growing in soft agar, and after 20 in vitro passages, cells of all lines were characterized with respect to their ability to form colonies in soft agar and their tumorigenicity in hamsters. While the studies showed that there are striking differences among the lines with respect to colony-forming ability, and real, though less striking differences in tumorigenicity, they failed to reveal any obvious differences between the groups of cell lines transformed by chymo- and chymo+ polyoma virus. Of 13 lines examined, all were found to express both middle and small polyoma T antigens, none express significant levels of large T antigen, and 11 express some form of what is probably a truncated large T antigen, the most common species having a molecular weight of 67000.     

Cooperation of p53 and polyoma virus middle T antigen in the transformation of primary rat embryo fibroblasts.

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.     

Loss of functional large T-antigen and free viral genomes from cells transformed in vitro by polyoma virus after passage in vivo as tumor cells.

We have analyzed the state, arrangement, and expression of polyoma viral DNA sequences in a number of in vitro-transformed Fischer rat cells before and after growth in vivo as tumour cells. When the in vitro lines used to induce the tumors contained only a single insert of viral sequences and did not produce either a full-size 100,000-dalton (100K) large T-antigen or free viral genomes, no differences in the above-mentioned properties were observed. By contrast, in vitro cell lines containing multiple inserts of viral sequences, a functional 100K large T-antigen, and free viral genome induced tumor cells which displayed a reduced number of inserts of viral sequences and which did not produce either a functional 100K large T-antigen or free viral genomes. All of the in vitro lines and their tumor cell derivatives expressed the polyoma virus 55K middle and 22K small T-antigen species. Possible mechanisms for the selection in vivo against cells containing a functional 100K large T-antigen and consequently free viral genomes are discussed.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

Characterization of polyoma viral DNA sequences in polyoma-induced hamster tumor cell lines

The polyoma (PY) viral DNA sequences present in a series of hamster tumor cell lines were evaluated using the blotting technique of Southern ((1975) J. Mol. Biol. 98, 503-517). Remarkably, no cell line contained an intact distal portion of the early gene region which encodes a portion of the large T antigen. All cell lines examined contained the PY DNA Bum I fragment which contains most of the genetic information encoding PY small and middle T antigens, as well as the origin of viral DNA replication. These results provide an explanation for our previous observation that PY virus-induced hamster tumors do not contain the large species of T antigen.     

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.