Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the ts A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were grown at the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpermissive temperature. All of the wild-type virus-transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. These results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristic of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells. 619 on August 1, 2020 by guest http://jvi.asm.org/ Downloaded from 620

The hepatitis B virus X protein (HBx) is a broadly acting transactivator implicated in the development of liver cancer. Recently, HBx has been reported to interact with several different cellular proteins, including our report of its binding to XAP-1, the human homolog of the simian repair protein UVDDB. In the present study, several HBx mutants were used to localize the minimal domain of HBx required for binding to XAP-1/UVDDB to amino acids 55 to 101. The normal function of XAP-1/UVDDB is thought to involve binding to damaged DNA, the first step in nucleotide excision repair (NER); therefore, we hypothesized that this interaction may affect the cell’s capacity to correct lesions in the genome. When tested in two independent assays that measure NER (unscheduled DNA synthesis and host cell reactivation), the expression of HBx significantly inhibited the ability of cells to repair damaged DNA. Under the assay conditions, HBx was expressed at a level similar to that previously observed during natural viral infection and was able to transactivate several target reporter genes. These results are consistent with a model in which HBx acts as a cofactor in hepatocarcinogenesis by preventing the cell from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, cancer. An adverse effect on cellular DNA repair processes suggests a new mechanism by which a tumor-associated virus might contribute to carcinogenesis.

The mechanism of action of hepatitis B virus (HBV) X protein in transcriptional transactivation and in tumorigenesis remains obscure. We have used the yeast two-hybrid system to identify a cellular protein that can interact with HBV X protein. This protein, designated X-associated protein 1 (XAP-1), is a human homolog of the UV-damaged DNA-binding protein (UV-DDB) recovered from a monkey cell cDNA library. UV-DDB is presumed to be involved in DNA repair. The interaction between X protein and XAP-1 protein was verified by immunoprecipitation of yeast cell lysates expressing both proteins and by in vitro mixing with X protein expressed as a glutathione S-transferase fusion protein and XAP-1 protein either in HeLa cell extracts or synthesized by in vitro translation. We speculate that the interaction of X protein with a DNA repair protein may recruit cellular proteins to repair the partially double-stranded HBV genome or may modify cellular transcription processes. An effect on the cellular DNA repair system may explain a cofactor role for HBV in liver cancer development.

No abstract

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [3S]methionineor 'Pi-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of -96,000 was detected in both subcellular fractions. Minor components of -68,000 and -56,000 with anti-T reactivity which labeled with [3S]methionine were also detected in both fractions from H-50 cells, as were components of -140,000 and -56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in 32Pi-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [3H]thymidine labeling, NADH-diaphorase activity, and Na+-K+-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen. 523 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from 524 SOULE AND BUTEL Al was added to each antigen-antibody reaction. After incubation for 5 min at 0°C, the immune complexes were washed three times with BDM by centrifugation at 2,400 x g for 10 min. Antigen was eluted from the immune complex by resuspending the final bacterial pellet in 50 pl of electrophoresis sample buffer (0.0625 M Tris [pH 6.8], 2% SDS, 2% ,-mercaptoethanol, 5% glycerin, and 0.005% bromophenol blue) and boiling for 3 min. Bacteria were removed by centrifugation at 17,000 x g for 10 min at 4°C. The supernatant was carefully removed and subjected to SDS-gel electrophoresis on discontinuous 12.5% SDS-polyacrylamide slab gels (14 by 12 by 0.15 cm) as previously described (16,19,39,40), with acrylamide-methylene bisacryl-J. VIROL.on July 6, 2020 by guest