An oligomeric form of simian virus 40 large T-antigen is immunologically related to the cellular tumor antigen p53.

The induction of tumors and cellular transformation mediated by polyomavirus requires the action of middle T antigen. Accordingly, we have begun to define the domains of the viral protein important for these processes to learn more about its site and mechanism of action. One of the domains of middle T antigen which is thought to be important for its function includes a stretch of acidic amino acids and a vicinal tyrosine residue (tyrosine 315), the major site of tyrosine phosphorylation in vitro. To determine whether these acidic amino acids and tyrosine 315 are required to maintain the transforming activity of middle T antigen, we constructed deletions within the DNA sequences encoding these amino acids and measured the capacity of the resulting mutants to transform Rat-1 cells in culture. This was accomplished by using in vitro mutagenesis techniques with molecularly cloned polyomavirus DNA. Seven mutants were isolated. Five of these proved incapable of transforming Rat-1 cells and were found to contain deletions which altered the reading frame for middle T antigen. However, two mutants, pPdl1-4 and pPdl2-7, retained the capacity to transform Rat-1 cells at high frequencies. The middle T antigen encoded by one of these mutants, pPdl1-4, lacks part of the acidic string of amino acids but not tyrosine 315 (amino acids 304 through 310 are deleted), whereas the middle T antigen encoded by the other mutant, pPdl2-7, lacks the entire acidic amino acid stretch as well as tyrosine 315 (amino acids 285 through 323 are deleted). Rat-1 cells transformed by one or the other mutant DNA displayed a fully transformed phenotype, including the capacity to form tumors in animals. These results prove that the major site of tyrosine phosphorylation in middle T antigen and the acidic amino acids which precede it are not essential for its transforming activity.     

Polyoma viral middle T-antigen is required for transformation.

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdl1-4 and PPd12-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdl1-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.     

Simian virus 40 origin DNA-binding domain on large T antigen.

Fifty variant forms of simian virus 40 (SV40) large T antigen bearing point, multiple point, deletion, or termination mutations within a region of the protein thought to be involved in DNA binding were tested for their ability to bind to SV40 origin DNA. A number of the mutant large T species including some with point mutations were unable to bind, whereas many were wild type in this activity. The clustering of the mutations that are defective in origin DNA binding both reported here and by others suggests a DNA-binding domain on large T maps between residues 139 and approximately 220, with a particularly sensitive sequence between amino acids 147 and 166. The results indicate that the domain is involved in binding to both site I and site II on SV40 DNA, but it remains unclear whether it is responsible for binding to cellular DNA. Since all the mutants retain the ability to transform Rat-1 cells, we conclude that the ability of large T to bind to SV40 origin DNA is not a prerequisite for its transforming activity.     

Site-directed mutagenesis of the simian virus 40 large T-antigen gene: replication-defective amino acid substitution mutants that retain the ability to induce morphological transformation.

We used a heteroduplex deletion loop mutagenesis procedure for directing sodium bisulfite-induced mutations to specific sites on viral or plasmid DNA to generate a series of SV40 large T-antigen point mutants. The mutations were directed to a region of the T-antigen gene, 0.5 map units, that is thought to be important for interaction of the protein with the viral origin of DNA replication. Of the 16 mutants reported here, 10 had lost the ability to replicate their DNA, and 3 others showed a reduced level of replication compared to wild type. All of the mutants tested were capable of transforming rat cells in culture by the dense focus assay. We conclude that the sequences of the early region around 0.5 map units are critical for the replication of viral DNA but not for the transformation function of T antigen.     

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.     

Recent progress in studies of polyomavirus tumour antigens

The polyomavirus tumour (T) antigens were originally identified by their reactivity with antisera from tumour-bearing animals. The primary structure of the three T-antigens has been established by combining the information from the nucleotide sequencing of DNA, RNA analysis, and peptide mapping. The functions of the T-antigens in productive infection and cellular transformation have largely been analysed by using virus mutants. The large T-antigen binds specifically to polyomavirus DNA. This binding is probably linked to the activity of the protein in the control of viral DNA and RNA synthesis. In addition, the large T-antigen has the ability to confer an unlimited growth potential to cells in culture. The middle T-antigen is a primary inducer of cellular transformation. The part of this protein that is located in the plasma membrane, is associated with a tyrosine kinase activity. The small T-antigen, finally, has not yet been studied extensively. However, small T-antigen has to be expressed to allow a complete productive infection cycle in mouse cells.     

The tumor antigens and the early functions of polyoma virus

Summary Polyoma virus (Py) tumor (T) antigens are the proteins specified by the early region of the viral genome. They are responsible for most biological effects caused by this oncogenic virus, i.e. induction of tumors, cell transformation and most of the virus-induced events observed in productive and transforming infection. By immunoprecipitation with antitumor serum followed by gel electrophoresis three major Py T-antigens have been characterized: large Tantigen (IT) with an apparent M_T of about 100 000, middle T-antigen (mT) of about 55 000 Mr and small T-antigen (sT) of about 23 000 Mr. In addition, there may exist one or more minor species by Py T-antigens. Analysis of the tryptic peptides showed that IT, mT and sT have a common N-terminal amino acid sequence, but differ from each other in the size and the sequence of the C-terminal part of the molecule as a consequence of different splicing of their mRNAs. With the nucleotide sequence of the Py genome being known, the coding regions for each of the Py T-antigens have been identified and consequently the amino acid sequence of IT, mT and sT was deduced. Cell fractionation experiments showed that the major part of 1T is located in the nucleus, mT was found in plasma membranes and sT is mainly present in the cytoplasm. Large T is a phosphoprotein and undergoes posttranslational modification. Two-dimensional gel electrophoresis of Py T-antigens revealed considerable charge heterogeneity particularly for mT and sT.All Py transformed cell lines analyzed contained mT and sT. Large T was not detected in virtually all Py transformed mouse cell lines and in about one third of Py transformed rat and hamster cell lines. Instead of 1T often new immunoreactive proteins were found which are probably truncated forms of 1T. These and other recent results suggest that IT is required neither for initiation nor for maintenance of cell transformation. For tumor induction in hamsters, similar conclusions were reached from analysis of Py T-antigens and viral DNA sequences in cell lines derived from tumors that had been induced either by virus or by viral DNA digested with various restriction enzymes. Experiments done with several deletion mutants indicated that mT is required for cell transformation by Py. In a protein kinase assay done in vitro with Py T-antigen immunoprecipitates, a kinase activity associated with Py mT was found which phosphorylates tyrosine residues mainly of mT and less frequently of 1T and of rat immunoglobulins. In all transformation defective mutants, kinase activity measured by this assay was absent or strongly reduced.In a concluding chapter I discuss the events occurring in wild-type virus and mutant infected cells trying to attribute specific functions to each of the three Py T-antigens. At least two functions are known for 1T, one is initiation of viral DNA replication, the other induces a mitotic response of the host cell, i.e. the events leading to and including host chromatin duplication. Middle T-antigen is certainly involved in cell transformation, possibly by its presence in the membrane. No function has been defined yet for sT. Since there are more virus-induced events observed in infected cells than Py T-antigens at least one of them must be a multifunctional protein.     

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.     

Definition of regions in human c-myc that are involved in transformation and nuclear localization.

To study the relationship between the primary structure of the c-myc protein and some of its functional properties, we made in-frame insertion and deletion mutants of the normal human c-myc coding domain that was expressed from a retroviral promoter-enhancer. We assessed the effects of these mutations on the ability of c-myc protein to cotransform normal rat embryo cells with a mutant ras gene, induce foci in a Rat-1-derived cell line (Rat-1a), and localize in nuclei. Using the cotransformation assay, we found two regions of the protein (amino acids 105 to 143 and 321 to 439) where integrity was critical: one region (amino acids 1 to 104) that tolerated insertion and small deletion mutations, but not large deletions, and another region (amino acids 144) to 320) that was largely dispensable. Comparison with regions that were important for transformation of Rat-1a cells revealed that some are essential for both activities, but others are important for only one or the other, suggesting that the two assays require different properties of the c-myc protein. Deletion of each of three regions of the c-myc protein (amino acids 106 to 143, 320 to 368, and 370 to 412) resulted in partial cytoplasmic localization, as determined by immunofluorescence or immunoprecipitation following subcellular fractionation. Some abnormally located proteins retained transforming activity; most proteins lacking transforming activity appeared to be normally located.     

Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants

We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated.     

Functional analysis of a simian virus 40 super T-antigen.

The SV3T3 C120 line of simian virus 40-transformed mouse cells synthesizes no large T-antigen of molecular weight 94,000 but instead a super T-antigen of molecular weight 145,000. In the accompanying paper (Lovett et al., J. Virol. 44:963-973, 1982), we showed that the integrated viral DNA segment SV3T3-20-K contains a perfect, in-phase, tandem duplication of 1.212 kilobases within the large T-antigen coding sequences. Our data suggested that this integrated template encodes mRNAs of 3.9 and 3.6 kilobases, the smaller of which directs the synthesis of the super T-antigen of molecular weight 145,000. We transfected the DNA segment SV3T3-20-K into nonpermissive rat cells and into TK- mouse L cells and analyzed the T-antigens and viral mRNAs in the transfectants; these data prove directly the coding assignments suggested previously. The super T-antigen retained the ability to induce morphological transformation, and may even transform better than the wild-type protein. It also retained the ability to bind to the cell-coded p53 protein. Transfection into permissive CV-1 cells showed that the super T-antigen encoded by SV3T3-20-K was incapable of initiating DNA replication at the viral origin. The duplication in SV3T3-20-K thus defines a mutation which separates the transformation and DNA replication functions of large T-antigen. We discuss why such mutations may be selected in transformed cells.     

Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential.

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.     

Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250.

Tyrosine residues of middle-T and tyrosine phosphorylation are thought to be important in the transformation of cultured rodent cells by polyomavirus. Of the potential tyrosine sites in the carboxyl-terminal half of middle-T, tyrosines 297, 315, and 322 have been studied previously, whereas tyrosine 250 has not. Two mutant plasmids, XD121 and pT250, encode polyomavirus middle-T species in which the tyrosine 250 residue is affected. XD121 is a deletion mutant in which the region encoding tyrosine 250, together with three adjacent amino acids, is deleted, whereas pT250 is a point mutant in which the tyrosine 250 codon has been converted to a phenylalanine codon. The plasmids were handicapped in transforming ability, as judged by focus formation on a monolayer of Rat-1 cells. Both demonstrated a reduction in the number of foci produced and a lag in the time of appearance of foci when compared with wild-type plasmid. The importance of residue 250 in this phenotype was indicated by the observation that plasmids containing multiple mutations proximal to the tyrosine 250 codon were wild type in their transforming ability. Furthermore, a revertant of pT250 (pT250-w.t.), which utilized the alternative tyrosine codon of TAC, was shown to regain full transforming activity. A combined-mutant plasmid, pTH, encodes a middle-T species in which both tyrosines 250 and 315 are converted to phenylalanine. This plasmid was totally defective in the transformation of rodent cells in a focus formation assay; however, it did impart a small measure of anchorage-independent growth when the encoded protein was expressed in NIH 3T3 cells. The in vitro kinase activity and pp60c-src association of the mutant middle-T antigens were examined. These assays demonstrated a reduction in phosphate acceptor activity for the middle-T species encoded by pT250 and pTH. Quantitative kinase assays showed that all of the tyrosine-mutant middle-T species, encoded by pAS131 (containing the tyrosine 315 codon-to-phenylalanine codon mutation), pT250, and pTH, were able to enhance pp60c-src kinase activity but only at levels which were intermediate and which reflected their transforming abilities relative to wild type.     

Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome

Isolated restriction endonuclease fragments of the herpes simplex virus type 1 (HSV-1) genome were introduced into hamster embryo cells to identify DNA sequences capable of transforming the cells with respect to acquisition of properties correlated with tumorigenicity. One of the fragments generated by cleavage of HSV-1 DNA with the restriction endonuclease Xba I was found to induce transformation at a frequency of about 10 colonies per quantity of fragment recovered from 1 μg of uncut DNA; fractions containing the other Xba I fragments failed to induce transformation reproducibly, although occasional colonies were detected. The fragment with transforming activity (Xba I-F) is 15.5 × 10⁶ daltons in molecular weight and is located between 0.30 and 0.45 map units on the HSV-1 genome. The Xba I-F transformants obtained were selected for their ability to replicate in low concentrations of serum; in addition, they were found to attain high saturation densities in the presence of 10% serum and to form colonies in semisolid medium. Moreover, the transformed cells produced at least one of the viral gene products (a membrane glycoprotein) encoded in the fragment used for transformation, indicating not only that viral DNA was incorporated into the cells, but also that viral genes were expressed.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.     

Homologous recombination between transfected DNAs.

An extensive analysis of the fate and structure of polyomavirus-plasmid recombinant molecules transfected into Rat-1 cells has revealed that the DNA often becomes integrated within transformed cell DNA in a head-to-tail tandem arrangement. This occurs independently of the replicative capacity of the transforming DNA and is facilitated by the use of large quantities of DNA during transfection. These observations have led us to suggest that head-to-tail tandems are formed by homologous recombination between transfected DNAs either before or after integration within cellular DNA. To test this hypothesis, we have measured the transforming activity of pairs of mutant, nontransforming, recombinant plasmid DNAs that carry different lesions in the transforming gene of polyomavirus. The results show that, although the individual mutant DNAs are incapable of transformation, transfection with pairs of mutant DNAs leads to the formation of transformed cells at high frequency. Moreover, there is a direct relationship between the distance between the lesions in pairs of mutant DNAs and their transforming activity. Finally, analyses of the structures of integrated recombinant plasmid DNAs and the viral proteins within independent transformed cells prove that recombination occurs between the mutant genomes to generate a wild-type transforming gene.