Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts (original) (raw)
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Journal of Virology
Puromycin was used to study the effect of the inhibition of protein synthesis on transformation of hamster cells (BHK21) by polyoma virus. The drug was used at a concentration (10-4 M) which caused in these cells a drastic but fully reversible inhibition of protein synthesis. A two-to threefold enhancement of transformation rate was obtained when the cells were exposed to puromycin for a period of 5 hr that started at the end of the virus adsorption period. No further enhancement was produced by prolonging puromycin treatment up to 13 hr after infection. The possibility that the observed effect on transformation rate could be mainly attributed to cell selection by puromycin was excluded. In addition, the relevance of a number of possible secondary effects of puromycin (inhibition of cell division, inhibition of deoxyribonucleic acid synthesis, etc.) was also ruled out. The effect of puromycin on transformation appeared to be dependent on the time (relative to infection) of addition of the drug. In fact, no transformation enhancement was observed when the cells were exposed to puromycin prior to infection or beyond the 10th hr after infection. Since another drug known to affect protein synthesis (p-fluorophenylalanine) was also shown to produce similar effects, it is suggested that transformation enhancement results from the inhibition of protein synthesis during a sensitive period closely following adsorption of the virus.
Molecular and Cellular Biology, 1982
Polyoma virus transforms, upon infection or DNA transfection, nonpermissive Fisher rat fibroblasts. Cloned viral DNA was deleted of sequences around the Bg/I site at nucleotide 86 by Bal31 nuclease treatment and then recloned in Escherichia coli. The extent of deletion for each mutant was then determined by DNA sequencing. Deletions included the early transcription control signals; others stretched into the N-terminal coding sequences of the viral tumor antigens. The transformation efficiency of 16 mutants was tested by transfecting rat fibroblasts. Expression of the T antigens was analyzed by immunofluorescence detection after transfection of rat fibroblasts, mouse secondary embryo cells, and HeLa cells. We found that the absence of the early transcription control sequences (TATA and CAAT boxes) did not significantly alter the transformation capacity of the virus. On the other hand, deletion of the initiator methionine ATG codon or further into the coding sequences did abolish the ...
Viral gene expression in polyoma virus-transformed rat cells and their cured revertants
Journal of Virology
We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variable amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infection; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's, but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended into flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. Si analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral DNA began 40 base pairs past the AAUAAA sequence at 25.8 map units, no readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region.
Journal of Virology
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 nuclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specific 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.
Journal of Virology
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 nuclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specific 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.
Proceedings of the National Academy of Sciences, 1983
Transfer into mouse and rat embryo fibroblasts in primary culture of cloned polyoma virus genes encoding only the large T protein led to the establishment of flat colonies in sparse subcultures at a frequency equal to that of transformation by wildtype virus. Cell lines could be derived from such colonies and maintained in culture for large numbers of generations without entering crisis. They exhibited a normal phenotype, by the criteria of growth on plastic to a low saturation density and of anchorage dependency. However, they required a lower serum concentration for growth than spontaneously established 3T3 cells. Similar results were obtained after transfer of recombinant DNA molecules encoding only the amino-terminal 40% of the large T protein, suggesting that this "immortalization" function corresponds to the activity of an amino-terminal domain of the protein.