Multiple forms of polyoma virus tumor antigens from infected and transformed cells.

At least three distinct forms of polyoma virus tumor antigens were isolated from productively infected and transformed hamster cells by immunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three T-antigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome.     

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.     

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.     

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.     

On the functional roles of simian virus 40 large and small T-antigen in the induction of a mitotic host response.

The early gene of wild-type (wt) SV40 specifies two related proteins, referred to as large (Mr 88,000) and small (Mr 19,000) T-antigen. Infection with wt SV40 of Go/G1-arrested monkey kidney and CV-1 cell cultures induced in virtually 100% of the cells T-antigen synthesis, followed by a mitotic reaction and the production of SV40 DNA. Parallel cultures were infected with SV40 deletion mutants that produce either no small T-antigen (d1883) or only trace amounts of a truncated form (d1891). Kinetics of synthesis and accumulation of large T-antigen was closely similar to that observed with wtSV40 whereas apparently only 50-60% of the cells participated in the mitotic reaction and the production of viral DNA. These results and those obtained from a comparative study on the abortive (transforming) infection in Go-arrested mouse tissue culture cells indicate that synthesis of large T-antigen alone is sufficient to trigger in 50-60% of the infected cells a mitotic reaction.     

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.     

Stability of polyoma DNA sequences and virus-coded proteins during tumor formation

To determine the stability of polyoma viral DNA in transformed rat cells during their growth in vivo, we compared the state and arrangement of polyoma virus DNA sequences in virus-transformed rat cell lines before and after their passage in vivo. In cell lines from 12 independent tumors induced by the inoculation of animals with three different transformed cell lines, we could detect no significant changes in the arrangement of viral DNA sequences associated with the in vivo passage of these cell lines. In 13 of 14 tumor cell lines examined, the pattern of polyoma virus tumor antigens, characterized by the presence of the polyoma virus large, middle, and small tumor antigens, was unchanged.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

Interactions of Polyoma and Mouse DNAs. IV. Time Course and Extent of Integration of Polyoma DNA into Mouse DNA During Lytic Infection

The time course of covalent binding of polyoma viral DNA to mouse DNA was followed in mouse embryo cells that had been grown prior to infection in the presence of 5-bromodeoxyuridine. Density-labeled (HL) mouse DNA was separated from free polyoma DNA by CsCl isopycnic centrifugation. Polyoma DNA sequences present in HL mouse DNA were detected by hybridization with radioactive cRNA synthesized in vitro. In reconstruction experiments, the limit of detection was found to be, on the average, about 0.5 genome equivalent (g.e.) of polyoma DNA per cell. To find conditions for the isolation of HL mouse DNA and for its complete separation from free polyoma DNA, cultures infected at 4 degrees C were used. HL mouse DNA extracted with sodium dodecyl sulfate and high salt concentrations (5 to 6 M CsCl) and then purified by three consecutive CsCl density gradient centrifugations was free from detectable amounts of polyoma DNA, whereas HL mouse DNA extracted with chloroform and phenol and purified in the same way always contained contaminating, noncovalently bound polyoma DNA. In lytically infected bromodeoxyuridine-prelabeled mouse embryo cultures, polyoma DNA bound to HL mouse DNA that had been extracted by the sodium dodecyl sulfate-CsCl procedure was first detected in small amounts (1 to 2 g.e. per cell) at 10 h after infection. In cultures incubated with medium containing thymidine (5 mug/ml), 4 to 6 g.e. of polyoma DNA per cell was detected at 14 and 18 h after infection. In these samples, practically all viral DNA was bound to high-molecular-weight HL mouse DNA. In cultures incubated with normal medium (no additions) and extracted between 17 and 20 h after infection, 20 to 350 g.e. of polyoma DNA per cell banded with HL mouse DNA. However, when DNA of one of these samples was subfractionated by sodium dodecyl sulfate-salt precipitation prior to isolation of HL mouse DNA, about 80% of the viral DNA banding at increased density was present in the low-molecular-weight DNA fraction. This observation suggests that in normal medium some progeny viral DNA of increased density was synthesized. Covalent binding of polyoma DNA to density-labeled mouse DNA was demonstrated by alkaline CsCl density gradient centrifugation: nearly equal amounts of polyoma DNA were found in the H and L strands, respectively, as is expected for linear integration of viral DNA. The results lead to the conclusions that (i) early polyoma mRNA is transcribed from free parental viral DNA; (ii) covalent linear integration is first detectable at the time when tumor (T)-antigen is synthesized; and (iii) only few copies (<10 g.e./cell) become integrated between 10 and 18 h after infection, i.e., during the period when cellular and viral DNA replication starts in individual cells.     

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

Multiplication of Polyoma Virus in Mouse-Hamster Somatic Hybrids: a Hybrid Cell Line Which Produces Viral Particles Containing Predominantly Host Deoxyribonucleic Acid

The multiplication of polyoma virus in a mouse-hamster (3T3 x BHK) somatic hybrid line (10A), which, although permissive for viral multiplication, produces very low amounts of virus, has been studied. In this cell line, the efficiency of productive infection is high, but the yield of infectious virus is on the order of 0.5% of that of 3T3 cells. The amount of viral deoxyribonucleic acid (DNA) synthesized by these cells upon infection is about 5% of that of 3T3 cells. An examination of the virus produced in hybrid 10A revealed that it was only one-tenth as infectious as the virus grown in 3T3. Although the viral DNA synthesized in the infected 10A cells is normal, the DNA extracted from purified virus grown in 10A consists of approximately 10% of normal, supercoiled polyoma DNA molecules and of approximately 90% linear DNA molecules with a sedimentation coefficient of 14 to 16S. These DNA molecules appear to be of cellular origin but contain a limited amount of viral DNA sequences. The host DNA-containing particles are not infectious but appear to possess some biological activity; they give rise to a weak complementation effect, and part of them are able to induce T-antigen synthesis. In addition, the host DNA present in these particles is predominantly that which has been synthesized after infection. The correlation between the block in viral DNA synthesis in this cell line and the abnormal encapsidation of host DNA is discussed.     

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.     

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.     

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.     

Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively.     

Loss of Polyoma Virus Infectivity as a Result of a Single Amino Acid Change in a Region of Polyoma Virus Large T-Antigen Which Has Extensive Amino Acid Homology with Simian Virus 40 Large T-Antigen

The polyoma virus (Py) transformed cell line 7axB, selected by in vivo passage of an in vitro transformed cell, contains an integrated tandem array of 2.4 genomes and produces the large, middle, and small Py T-antigen species, with molecular weights of 100,000, 55,000, and 22,000, respectively (Hayday et al., J. Virol. 44:67-77, 1982; Lania et al., Cold Spring Harbor Symp. Quant. Biol. 44:597-603, 1980). The integrated viral and adjacent host DNA sequences have been molecularly cloned as three EcoRI fragments (Hayday et al.). One of these fragments (7B-M), derived from within the tandem viral sequences, is equivalent to an EcoRI viral linear molecule. Fragment 7B-M has been found to be transformation competent but incapable of producing infectious virus after DNA transfection (Hayday et al.). By constructing chimerae between 7B-M and Py DNA and by direct DNA sequencing, the mutation responsible for the loss of infectivity has been located to a single base change (adenine to guanine) at nucleotide 2503. This results in a conversion of an aspartic acid to a glycine in the C-terminal region of the Py large T-antigen but does not appear to affect the binding of the Py large T-antigen to Py DNA at the putative DNA replication and autoregulation binding sites. The mutation is located within a 21-amino acid homology region shared by the simian virus 40 large T-antigen (Friedmann et al., Cell 17:715-724, 1979). These results suggest that the mutation in the 7axB large T-antigen may be involved in the active site of the protein for DNA replication.     

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells.

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.