Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.