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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Interspecies-, species- and type-specific T antigenic determinants of human papovaviruses (JC and BK) and of Simian virus 40.

Immunofluorescence tests, absorption studies and quantitative analysis by a very sensitive 51Cr microcomplement fixation (CF) technique were used to define the degree of relatedness between the tumor (T) antigens induced by human papovaviruses, strain JC and BK, with simian virus 40(SV40) and mouse polyoma virus (PyV). Antisera against JCV, BKV, SV40 and PyV T were raised in tumor-bearing hamsters. The data obtained indicate that T antigens of JCV, BKV and SV40 possess various subspecificities which can be distinguished and looked upon as interspecies-, species- and type-specific antigenic determinants.

Similar cell surface antigens on hamster cells transformed by different papovaviruses.

Transformed baby hamster kidney (BHK) cells were tested for surface antigens by an immunocytoadhesion method. The cells were sensitized with rabbit antisera to cell clones transformed by polyoma or by BK virus and then rosetted with erythrocytes coated with antibody to rabbit immunoglobulin. These antisera detected common antigens on BHK cells transformed by either of three papovaviruses, polyoma, BK, or SV40, but apparently not on normal BHK cells.

Somatic mutation as the basis for malignant transformation of BHK cells by chemical carcinogens.

The chemical induction of malignant transformation in BHK cells seems to result from a somatic mutation. Stable transformants, whose frequency is significantly increased by mutagenic carcinogens, can revert to normal and often display temperature-restricted phenotypes indicative of an altered gene product.

Occurrence of BK virus DNA in DNA obtained from certain human tumors.

DNA sequences from BK virus have been detected by measurements of reassociation kinetics in DNA preparations isolated from five of 12 human tumor tissues and three of four human tumor cell lines; no DNA sequences from BK virus could be found using the same assay in DNA samples obtained from nontumor tissues removed from various patients. Whether or not this association is trivial, indicative of infection by BK virus, or correlative with the tumor state must await the testing of additional samples of normal and tumor tissues as well as a definition of the physical state of the genome of BK virus.