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...

The 55-kDa gene product from subgroup C adenovirus type 5 (Ad5) early region 1 (E1B-55kDa) plays a central role in the oncogenic transformation of primary rodent cells primarily by inactivating transcriptional and presumably other functional properties of the tumor suppressor protein p53. We have previously shown that Ad5 E1B-55kDa possesses a leucine-rich nuclear export signal (NES), which confers rapid nucleocytoplasmic shuttling via the CRM1-dependent export pathway. In this study we report that an export-deficient mutant of the viral protein (E1B-NES) substantially enhances focus formation of primary baby rat kidney cells in combination with Ad E1A. Transformed rat cells stably expressing the E1B-NES protein exhibited increased tumorigenicity and accelerated tumor growth in nude mice compared to transformants containing the wild-type E1B product. This 'gain of function' correlated with enhanced inhibition of p53 transactivation in transient reporter assays and the accumulation of the mutant protein and p53 in several dotlike subnuclear aggregates. Interestingly, these structures also contained a large fraction of cellular promyelocytic leukemia protein (PML), a known regulator of p53. These data indicate that E1B-NES promotes oncogenic transformation by combinatorial mechanisms that involve modulation of p53 in the context of PML nuclear bodies. In sum, these results extend our previous observation that inhibition of PML activities by E1B-55kDa is required for efficient focus formation and provide further support for the view that blocking p53 transcriptional functions is the principal mechanism by which the Ad protein contributes to complete cell transformation in conjunction with Ad E1A.

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