Persistence of type 5 adenovirus DNA in cells transformed by temperature-sensitive mutant, H5ts125.

Abstract

The characteristic of H5ts125, a temperaturesensitive DNA-minus mutant, to transform 3 to 8 times more rat embryo cells than wild-type 5 adenovirus was correlated with the persistence of an increased proportion of the viral genome in cells independently transformed at the nonpermissive (39.50) or semipermissive (360) temperature. Reassociation (3)(4)(5)(6). Hence, the isolation and characterization of temperature-sensitive mutants of adenoviruses (7-10) suggested that they, too, may be useful to identify the critical gene(s) involved in, and to detect the gene product(s) essential for, transformation. In contrast to our expectation from data obtained with Rous sarcoma (1) and SV40 (2) viruses, however, an adenovirus mutant defective in DNA replication (5), H5ts125, was discovered that transformed rat embryo cells at frequencies 3 to 8 times greater than wild-type 5 adenovirus at the nonpermissive (39.50) and semipermissive (36°) temperatures (11,12). This finding implies that a specific viral gene product affects transformation, perhaps by modulating against it, so that, when the viral protein is nonfunctional or functions aberrantly, transformation is less restricted. A single-strand specific DNA-binding protein has been identified as the viral protein encoded in the ts125-mutated gene (13)

Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration

“…Cot analyses of a number of cell lines transformed by H5ts125, H5tslO7, and Ad5 WT confirmed previous findings (23) indicating that expression of a defective DNA-binding protein in adenovirus mutants often results in persistence of all or most of the entire viral genome in the transformed host cell, whereas Ad5 WTtransformed cells usually contain only the lefthand end of the viral DNA molecule (23,26). Data obtained by the Southern blotting tech- Analysis of HindIII digests of DNA from rat embryo fibroblasts transformed with Ad5 WT and H5ts125.…”

“…Hence, none of the cell lines transformed by Ad5 WT studied, including those in previously published results (23), contained the entire viral genome. In addition, two cell lines transformed by H5ts125 at the permissive temperature (320C), like those studied earlier (23), contained only the left-hand end of the adenovirus genome. In contrast, the entire viral genome was present in approximately 50% of the cell lines transformed by H5ts125 or H5tslO7 at the semipermissive or nonpermissive temperature: some of the cell lines (e.g., 83) contained approximately one copy, whereas A18 contained four to five copies of the viral genome ( Table 1).…”

“…When semipermissive rat embryo fibroblasts are transformed by adenovirus type 2 or 5 (Ad2 or Ad5), about 15% of the viral genome, representing the left end, usually persists in the transforned cell, although on occasion a larger proportion of the viral DNA is present (14,15,23,26). However, when cells are transformed with the DNA-binding protein mutant H5ts125, which transforms rat embryo fibroblasts with a threeto eight-times-higher frequency than wild-type Ad5 (Ad5 WT) at the nonpermissive (39.5°C) or semipernissive (3600) temperature (17), cells often contain one or more copies of the entire adenovirus genone (23). These findings, however, do not describe the physical state of the viral DNA in H5ts125-transformed cells.…”

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth

“…This suggests that viral DNA replication is not required for integration, in agreement with results reported previously for Adl2 in nonpermissive BHK cells (9), and the difference (roughly 50-fold) in transformation frequency at 38.5°C between Ad5 ts125 and AdS ts36 (15, 39) is not due to a similar difference in integration frequency. These experiments do nQt rule out a possible difference in the viral DNA sequences integrated by the two mutants (25). However, since the left-hand 7% of the genome contains the only sequences required for transformation and there is no correlation between any additional sequences that may be integrated and the tumorigenicity of the transformants (7, 20), we believe that variation in the sequences integrated is unlikely to be important in either transformation or tumorigenicity.…”

“…In vitro transformation by an avian adenovirus (CELO). I. Hamster-embryo fibroblastic cultures.VOL 25,. 1978 on September 28, 2020 by guest http://jvi.asm.org/ Downloaded from…”