Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orfb protein

Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene pr oduct displays features of a viral oncoprotein, It initiates focal transfor mation of primary rat cells in cooperation with Ad5 El genes and confers mu ltiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growt h in nude mice. It has been reported that E4orf6 binds to p53 and, in the p resence of the Ad5 E1B-S5kDa protein, antagonizes p53 stability by targetin g the tumor suppressor protein for active degradation. In the present study , we performed a comprehensive mutant analysis to assign transforming funct ions of E4orf6 to distinct regions within the viral polypeptide and to anal yze a possible correlation between E40rf6-dependent p53 degradation and onc ogenesis. Our results show that p53 destabilization maps to multiple region s within both amino- and carboxy-terminal parts of the viral protein and wi dely cosegregates with E4orf6-dependent acceleration of tumor growth, indic ating that both effects are related. In contrast, promotion of focus format ion and morphological transformation require only a carboxyterminal segment of the E4 protein. Thus, these effects are completely independent of p53 s tability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to th e oncogenic potential of Ads E4orf6. Although genetically separable, both a ctivities are Largely mediated through a novel highly conserved, cysteine-r ich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein.