MUTATIONAL ANALYSIS OF THE TRANSFORMING AND APOPTOSIS SUPPRESSION ACTIVITIES OF THE ADENOVIRUS E1B-175R PROTEIN

The role of the adenovirus-2 E1B 19-kDa (175R) T antigen in E1a-cooperative transformation was determined by cotransfection of plasmids expressing E1A or E1B 175R T antigens into primary rat kidney (BRK) cells. Transformed cells were selected by virtue of their resistance to the antibiotic Geneticin (G418) conferred by neo gene co-expression from plasmids coding for 175R. 175R cooperated efficiently with genomic E1a and specifically with the 289R protein coded by the 13S mRNA in the transformation of primary BRK cells. Mutational analysis of the 175R protein revealed that the N terminus and the C-terminal 30 amino acids are not essential for E1a-cooperative transformation. Several conserved sequences located in the middle of the 175R protein are essential for transformation. The effect of various mutants to suppress apoptosis (programmed cell death) induced by an anti-cancer agent, cisplatin, was examined in cells producing the E1A and E1B 175R proteins. Apoptosis was measured by flow cytometric analysis and indicates that the 175R protein efficiently prevents cisplatin-induced apoptosis. This suggests that the 175R function involved in transformation segregates with its ability to suppress cisplatin-induced apoptosis.