Free viral DNA in BK virus-induced hamster tumor cells

The biological properties of nine clonal lines of BK virus-induced hamster tumor cells were studied. All clonal lines were oncogenic and showed an enhanced ability to form colonies in semisolid medium. The cells of each clonal line contained T antigen; no virus could be rescued from any of the clonal lines. The number of viral DNA copies was determined in three of the clonal lines and varied from 10 to 20 copies per diploid amount of cell DNA. The state of the viral genome was studied in these lines, and the great majority of the viral DNA molecules appeared to be present as free (nonintegrated) molecules. At least six length classes of free defective BK virus DNA molecules, which all lacked a part of the late region of the genome, were detected in these cells. Three of the six length classes of BK virus DNA molecules acquired a TaqI recognition site, which suggested substitution of cellular DNA.     

BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes

Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host.     

Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.     

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.     

Amplification mediated by polyomavirus large T antigen defective in replication.

The polyomavirus large T antigen promotes homologous recombination at high rates when expressed in rat cells carrying the viral replication origin and two repeats of viral DNA sequences stably integrated into the cellular genome. Recombination consists of both reciprocal and nonreciprocal events and is promoted by mutants defective in the initiation of viral DNA synthesis (L. St-Onge, L. Bouchard, and M. Bastin, J. Virol. 67:1788-1795, 1993). We have extended our studies to a rat cell line undergoing amplification of the viral insert. We show that large T antigen promotes amplification independently of its replicative function but that its origin-specific DNA binding activity is not sufficient to promote homologous recombination.     

Complete nucleotide sequence of polyomavirus SA12

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.     

Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences.

Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal antibodies. The DNA fragments in the immunoprecipitates were cloned in plasmid vectors. Four plasmid clones were selected for a detailed investigation of the inserted mouse DNA fragments. Nucleotide sequencing and DNase I footprint experiments showed that T antigen binds to sites in these fragments consisting of two tandemly oriented G(A)AGGC pentamers separated by AT rich spacers of different lengths. The cellular binding sites are very similar in their architecture to the SV40-DNA binding site I. The isolated cellular DNA fragments with T antigen binding sites occur only once or a few times in the mouse genome. Our data help to further define the structure of T antigen's DNA binding sites. The genetic functions of the isolated cellular DNA elements are not known.     

A mathematical model of homologous recombination in cultured cells.

This work presents a model describing the rate of recombination between homologous segments of DNA stably integrated into the genome of cultured cells. The model has been applied to rat cell lines carrying the polyomavirus middle T oncogene and a functional origin of viral DNA replication. Introduction of the gene coding for the polyoma large T antigen or the SV40 large T antigen into cells by DNA transfection promotes homologous recombination in the resident viral inserts with rates varying between 0.1 x 10(-3) and 3.7 x 10(-1) per cell generation.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

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.     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts.

Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild-type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication.     

Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome

Pulse-labeled simian virus 40 (SV40) chromatin as well as uniformly labeled viral chromatin are immunoprecipitable by an SV40-specific tumor antiserum and therefore contain bound tumor antigen (T antigen). Single-stranded calf thymus DNA, immobilized on cellulose, competes effectively for T antigen binding with uniformly labeled nonreplicating, but not with pulse-labeled replicating, chromatin. Furthermore, T antigen dissociates in 0.5 M NaCl from nonreplicating chromatin and from purified SV40 DNA, whereas most T antigen remains associated with replicating chromatin even in the presence of 1.2 to 1.5 M NaCl. We used filtration through DNA-cellulose columns and treatment with high salt to prepare pulse-labeled immunoreactive viral chromatin. The viral DNA was digested before, and in other experiments after, immunoprecipitation with the restriction endonuclease HindIII. We found that SV40 DNA sequences, most probably representing the entire genome, remain in the immunoprecipitate after HindIII digestion, indicating an association of T antigen with origin-distal sections of replicating viral DNA. The results suggest that T antigen in replicating chromatin may be bound to regions close to replicating points. We performed control experiments with in vitro-formed complexes of T antigen and SV40 DNA. When these complexes were immunoprecipitated and HindIII digested we found, in agreement with previous studies, that only the origin containing the HindIII C fragment carried bound T antigen.     

New properties of simian virus 40 large T antigen

An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40. The 8K T antigen was weakly labeled in vivo with [35S]methionine or 32Pi. A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen. The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen. Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin. The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences. Antibodies against large T antigen interacted with native recA protein.     

Uniform representation of the human papovavirus BK genome in transformed hamster cells.

The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R-HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented.