Absence of BK virus sequences in transformed hamster cells transfected by human tumor DNA.

In an attempt to gain insight into the mechanism of oncogenic transformation by BK virus (BKV), a human papovavirus, we have probed for BKV sequences in transformed hamster cells in which oncogenic transformation had occurred as a result of transfection by human tumor DNA positive for BKV sequences. Even though the sources of the transfecting DNA contained BKV sequences, the transformed hamster cells which arose from the transfection for the most part did not retain BKV sequences. In only one barely detectable case was BKV-specific DNA found associated with chromosomal DNA, and in only a small minority of the transformed cells was BKV DNA detected in the Hirt supernatant, indicating an episomal configuration. Even in these few cases where BKV sequences were present in an episomal form, altered migration on gels of some BKV-positive bands (compared to bands derived from cloned viral DNA) suggested deletions and rearrangements of BKV DNA. We employed several different probe methodologies for these studies, including nick-translation, random primer and a non-isotopic biotinylated probe which gave a sensitivity that could detect better than 0.01 copy of viral genome per diploid cell. We conclude that transformation by transfection with human tumor DNA does not require persistence of the BKV viral genome, suggesting that either BKV virus was irrelevant to original oncogenesis, in analogy with models proposed by others for herpesvirus oncogenesis.

Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells.

A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft-agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium.

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)

Transformation by purified early genes of simian virus 40.

A rapid soft-agar assay using baby hamster kidney (BHK21 cl.13) cells has been developed to establish the functional roles for the large T and small t antigens of SV40 in transformation. Plasmids expressing either large T or small t antigens of SV40 have also been constructed and these plasmids have been used separately or in combination for transformation. A large T clone, pD3-05, containing a deletion in the small t-specific coding region [0.584-0.54 map units (mu)], transformed a low-background subclone of baby hamster kidney (BHK21 cl.13) cell line and F111 rat fibroblasts to anchorage independence at a low level (10-20 and 1%, respectively, of an early region clone from wild type [WT], pW2). A WT-derived small t clone, pW2-t, containing a deletion in the large T-specific coding region (0.373-0.169 mu), did not transform F111 cells, but transformed BHK21 cells at a very low level (about 2% of pW2). Another WT-derived small t clone, pW2-t/B1, containing a larger deletion in the large T-specific coding region (0.512-0.169 mu), did not transform either BHK21 or F111 cells. However, cotransformation with pD3-05 clone and pW2-t or pW2-t/B1 clone increased the frequency of transformation to about the same level as that of pW2. The ability of the small t clones to enhance the transformation efficiency of the large T clone was not due to recombination between the two plasmids, since cotransformation with pD3-05 and a small t clone without the polyadenylation [poly(A)] signal sequence from WT, pW-t8, did not increase the frequency of transformation. When the frequency of transformation was determined by the focus assay using F111 cells, pD3-05 transformed as well as pW2. Also, cotransformation with pD3-05 and pW2-t/B1 did not increase the frequency of focus formation. Therefore, the small t antigen was not required for this morphological transformation.

Multiple origins of the complementary defective genomes of RF and origin proximal sequences of GS, two human papovavirus isolates.

It has previously been shown that the genome of RF virus, a variant of the human papovavirus, BK, consists of two DNA species, one (R1a) with a deletion corresponding to the early and the other (R2) with a deletion corresponding to the late region of BKV (A. Pater, M.M. Pater, and G. di Mayorca (1980). J. Virol. 36, 480-487; A. Pater, M. M. Pater, R. M. Dougherty, and G. di Mayorca (1981a). Virology 113, 86-94). In this report transfection experiments are used to show that these DNA species are individually defective for infection and that both DNA molecules are required simultaneously for the infection of human embryonic kidney (HEK) cells. DNA fragments containing the origin of replication in each of the DNA species are analyzed to show that R1a contains three and R2 contains two origins of replication. In addition, several changes in the repeat region proximal to the origin of replication are observed. The changes involve deletions and insertions. Examination of the deleted junctions most often reveals involvement of short stretches of repeated sequences (hot spots) in recombination. Another observed change is the insertion into R2 of a 63-bp sequence which contains no homology to either BK or SV40 DNA. This insertion is into the late-promoter region of this late-region coding DNA and appears to replace a poor "TATA" box in BK wild type with a better TATA box with the correct spacing from the "CAAT" box. A 304-bp fragment containing the origin of replication, early and late promoters, and the repeat units proximal to the origin of replication of GS, another variant of human BKV papovaviruses has also been sequenced. Several rearrangements, including deletions and insertions in the repeat region, are observed. Moreover, when homologous regions of this virus are compared to that of BKV, five base changes are detected, one of which is in the 23-bp origin region. This base change gives this 23-bp palindrome of GS a perfect two-fold rotational symmetry.

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.

Chemical carcinogens transform BHK cells by inducing a recessive mutation.

Treatment of BHK cells with mutagenic carcinogens induced neoplastic transformation in a single step. This transformation displayed the characteristics expected for a recessive mutation. Increasing doses of carcinogens induced transformants with kinetics similar to the kinetics with which they induced 6-thioguanine-resistant or ouabain-resistant mutants in the same population of cells. Transformants with temperature-restricted phenotypes were easily induced by carcinogens which cause mutations by base changes, but when ICR frameshift mutagens were used, the proportion of temperature-limited transformants was inversely related to the frequency with which a particular mutagen induced frameshift mutations. In hybrids between pseudodiploid isogenic strains of normal and transformed BHK cells, transformation was expressed as a dominant trait when the transformed parent was induced by a papovavirus, but was suppressed as a recessive trait when the transformed parent arose spontaneously or was chemically induced. Segregation of transformation was observed upon growth of suppressed normal hybrids, and the transformed phenotype which was reexpressed was in most cases characteristics of the original transformed parent.

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.

Genome analysis of MG virus, a human papovavirus.

The genome of MG virus, a variant of the human papovavirus BK virus, consists of two molecules, M1 and M2, M1 and M2 have deletions which correspond to 0.33 to 0.55 map unit and 0.77 to 0.85 map unit, respectively, from the EcoRI site on the BK virus genome. Restriction enzyme analysis of the DNAs of these viruses revealed many alterations in both DNA species. Both M1 and M2 DNAs have three recognition sites for EcoRI. M1 has a single recognition site for HindIII and five sites for PvuII. M2 has a single recognition site for PuvII and three sites for HindIII. The sites of these and several other restriction enzymes on each DNA molecule were mapped after the cloning of M1 and M2 DNAs into pBR322 at their unique HindIII and PvuII sites, respectively.

Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation.

Thirty-six of 40 rat cell clones transformed to anchorage independence at low multiplicity of infection by simian virus 40 tsA58 were heat sensitive for continued expression of the transformed phenotype. tsA1499 is an 81-base-pair deletion at 21 map units which is like tsA58 in that it is also heat sensitive for lytic growth, belongs to the A complementation group, and produces rat cell transformants which contain a thermolabile T antigen. Unlike tsA58, however, tsA1499 generated rat cell transformants efficiently at the temperature at which it was lytically defective, and 10 of 17 clones transformed by tsA1499 were cold rather than heat sensitive for the continued maintenance of the transformed phenotype. The lytic and transforming activities of the A region thus appeared to function independently in mutant tsA1499.

Arrangement of the genome of the human papovavirus RF virus.

DNA from plaque-purified RF virus, a variant of BK virus, was found to contain two species of molecules. Hybridization of each DNA species to the fragments of BK virus DNA revealed that one species had a deletion corresponding to at least 50% of the late region and the other had a deletion corresponding to at least 40% of the early region of BK virus DNA. Analysis by cleavage of each RF virus DNA species with restriction endonucleases EcoRI, HindIII, AvaII, and PvuII, when compared with BK virus DNA, revealed that the size and number of fragments were different. These results suggest the loss of some restriction sites and the appearance of new sites, probably as a result of base changes in each RF virus DNA species. Furthermore, analysis of the restriction map of each DNA molecule revealed in insertion(s) in both DNA species.

Comparative analysis of GS and BK virus genomes.

Analysis of heteroduplexing between the genomes of GS virus, a BK-group virus, and the prototype BK virus revealed one region of nonhomology. Further analysis by cleavage of viral DNA with the restriction endonucleases EcoRI, HindIII, and HaeIII revealed that base changes in the GS virus genome spanned 0.6 to 0.7 map unit from the EcoRI site. Large T and small t antigens of GS virus appear to be similar in size to the BK virus antigens.

Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level.

New region of the simian virus 40 genome required for efficient viral transformation.

Viable mutants of simian virus 40 with deletions in three regions of the virus genome (map coordinates 0.21-0.17, 0.59-0.54, and 0.67-0.74) have been tested for their ability to transform rat fibroblasts to anchorage independence. Only those mutants whose deletions occur between 0.59 and 0.55 in the proximal part of the early region are defective in transforming ability. The most severely defective of these transform with less than one-hundredth the efficiency of wild type. They retain their defect when tested in Chinese hamster lung cells and when infection is initiated with viral DNA instead of intact virions. Complementation for transformation can be observed between these transformation-defective deletions and a simian virus 40 temperature-sensitive A mutant.