The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a gt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wildtype, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo, with the PDZ domain-containing protein hDlg͞SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domainbinding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.Human adenoviruses are organized into six subgroups, A through F, and in people cause primarily respiratory, gastrointestinal, and eye infections (1). After infection of rodents, however, subgroup A and B and some subgroup D adenoviruses are tumorigenic. In animals, subgroup A and B adenoviruses produce undifferentiated sarcomas at the site of virus injection, and the viral E1 region, consisting of the E1A and E1B genes, is both necessary and sufficient for this tumorigenicity. The transforming potentials of the nuclear E1A and E1B oncoproteins derive, at least in part, from an ability to complex with and inactivate the cellular tumor suppressor proteins pRB and p53, respectively (2).Compared with the subgroup A and B adenoviruses, subgroup D adenovirus type 9 (Ad9) is unique in eliciting only estrogen-dependent mammary tumors in female rats (3) and requiring the viral E4 region ORF1 gene (9ORF1) for oncogenicity (4, 5). The 9ORF1 gene codes for a 14-kDa cytoplasmic transforming protein (5, 6), and three separate regions of this viral polypeptide are important for transforming potential in cells (7), including a C-terminal region that mediates direct binding to several unidentified cellular proteins (p220, p180, p160, p155, p140͞p130; R.S.W. and R.T.J., unpublished work). To reveal the molecular mechanisms of the novel 9ORF1 oncoprotein, we sought to identify these 9ORF1-associated cel...