The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAFII31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAFII31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAFII31 but nevertheless inhibits the p53-TAFII31 interaction.

No abstract

We previously have shown that adenovirus type 5 E4orf4 protein associates with protein phosphatase 2A (PP2A) and induces apoptosis in transformed cells in a p53-independent manner. Here we show that the interaction between E4orf4 and PP2A is required for induction of apoptosis by the viral protein. This conclusion is supported by a mutation analysis of E4orf4 protein, showing a correlation between the ability to bind PP2A and to induce apoptosis, and by the observation that transfection of an antisense construct of the PP2A-B55 subunit reduces expression of the PP2A-B55 subunit and inhibits induction of apoptosis by E4orf4, but not by p53. The mutant analysis also indicates that even a low level of interaction with PP2A is sufficient to initiate the E4orf4 apoptotic pathway. In addition, E4orf4 inhibits cellular transformation by various oncogenes, and this function is coupled to its ability to induce apoptosis. Furthermore, expression of oncogenes in primary cell cultures sensitizes these cells to induction of apoptosis by E4orf4. Our results suggest that E4orf4 is a potentially useful tool for cancer gene therapy.

SUMO-1 is a small ubiquitin-related modifier protein that is covalently linked to many cellular and viral protein targets. Modification by SUMO-1 is proposed to play a role in protein targeting and͞or stability. We show here that adenovirus type 5 early region 1B 55-kDa (E1B-55kDa) oncoprotein can be covalently modified by SUMO-1 in vivo through a major attachment site comprising a single lysine residue at amino acid position 104. The sequence surrounding this lysine matches the proposed ⌿KxE consensus motif required for SUMO-1 conjugation. A single mutation (K104R) that abolishes SUMOylation of E1B-55kDa dramatically reduces the ability of the adenovirus type 5 protein to transform primary baby rat kidney cells in cooperation with E1A and to inhibit p53-mediated transactivation. Overexpression of SUMO-1 in adenovirus type 5 E1A͞E1B-55kDa-transformed baby rat kidney cells causes the relocalization of E1B-55kDa from the cytoplasm to the nucleus, where it accumulates with SUMO-1 in dot-or track-like structures. Significantly, when SUMO-1 is ectopically expressed in transformed rat cells no effect on the cytoplasmic localization of the E1B-K104R mutant protein is observed. Our results demonstrate that SUMO-1 modification is required for transformation by adenovirus type 5 E1B-55kDa and provide further evidence for the idea that this posttranslational modification plays a role in protein targeting to specific subcellular sites. T he 55-kDa phosphoprotein encoded in early region 1B (E1B-55kDa) from adenovirus type 5 (Ad5) is required for efficient viral DNA replication, selective viral late mRNA transport to the cytoplasm, and shut-off of host cell protein synthesis in productively infected cells (reviewed in ref. 1). In addition, the Ad protein provides functions for complete oncogenic transformation of mammalian cells in cooperation with Ad E1A (2). During the past few years it has been well established that the transforming potential of E1B-55kDa correlates with its ability to act as a direct transcriptional repressor targeted to p53-responsive promoters by binding to the tumor suppressor protein (3, 4). Considerable evidence suggests that these activities antagonize p53-induced apoptosis (5) and͞or cell cycle arrest (6). The regions required for transformation map to several segments in the Ad protein, including the p53-binding domains located around amino acid position 180 (Fig. 1A) and in the central part (4,7,8), plus two segments at the carboxy terminus that mediate inhibition of p53-dependent and p53-independent transactivation (5, 9, 10). Although the mechanism by which E1B-55kDa blocks transcription is still unclear, recent data suggest that its silencing activity requires a cellular corepressor that copurifies with RNA polymerase II (11) as well as interaction with cellular factors known to be involved in transcriptional repression such as histone deacetylase 1 (HDAC1) and mSin3A (12). Furthermore, consistent with its role in blocking p53-mediated transactivation, the Ad protein inhibits p53 acetylation b...