Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

Abstract

Previous studies have shown that the adenovirus type 5 (Ad5) E4orf6 gene product displays features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 E1 genes and confers multiple additional transformed properties on E1-expressing cells, including profound morphological alterations and dramatically accelerated tumor growth in nude mice. It has been reported that E4orf6 binds to p53 and, in the presence of the Ad5 E1B-55kDa protein, antagonizes p53 stability by targeting the tumor suppressor protein for active degradation. In the present study, we performed a comprehensive mutant analysis to assign transforming functions of E4orf6 to distinct regions within the viral polypeptide and to analyze a possible correlation between E4orf6-dependent p53 degradation and oncogenesis. Our results show that p53 destabilization maps to multiple regions within both amino-and carboxy-terminal parts of the viral protein and widely cosegregates with E4orf6-dependent acceleration of tumor growth, indicating that both effects are related. In contrast, promotion of focus formation and morphological transformation require only a carboxyterminal segment of the E4 protein. Thus, these effects are completely independent of p53 stability, but may involve other interactions with the tumor suppressor. Our results demonstrate that at least two distinct activities contribute to the oncogenic potential of Ad5 E4orf6. Although genetically separable, both activities are largely mediated through a novel highly conserved, cysteine-rich motif and a recently described arginine-faced amphipathic alpha helix, which resides within a carboxy-terminal "oncodomain" of the viral protein.

Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex

“…2C, Adp53KEEK) were readily degraded. As shown previously (51,53,63,78), such degradation required the expression of both viral proteins. Using the adenovirus vectors described for Fig.…”

Ovine atadenovirus: a review of its biology, biosafety profile and application as a gene delivery vector

“…LH3, although substantially shorter, shows homology with the mastadenovirus E1B 55 kDa protein that was identified by a BLAST search. This protein has an important role in cellular transformation and in the nuclear export of mRNA 23 (and references therein). E1B 55 kDa and E4 34 kDa proteins associate in a multi‐subunit complex that catalyses the ubiquitination and degradation of p53 21 , 24 .…”

“…The amino acid motif HCHCxxPGSLQC was identified by comparisons between OAdV7 E42 and E43 and mastadenovirus E4 34 kDa proteins 7 . Related motifs were subsequently found in chicken embryo lethal orphan (CELO) strain (avian adenovirus) orf14 and AO7, a mammalian RING finger protein that has been demonstrated to mediate binding and ubiquitination of proteins 23 , 28 . It is therefore tempting to speculate that E42 and E43 may form part of complexes that regulate certain proteins via ubiquitination pathways.…”

Xenogenic Adenoviral Vectors

“…For the aviadenoviruses, ORFs with homologies to dUTPase and the REP protein of adeno-associated virus have been found [23] but there are distinct differences between FAdVl and FAdV8 with three of eight ORFs in the left end being unique to FAdVS [74]. It was also reported recently [83] that the cysteines and several other residues in the conserved sequence motif of E4 ORF6 are conserved in FAdVl ORF14, which lies near the left end of the genome [23].…”

“…However, the E4 regions of human and animal mastadenoviruses vary in sequence and complexity. Homology with HAdV5 ORF6 is always evident, especially in a cysteine-rich motif [22] that is thought to mediate ORF6/p53 interaction [83]. Furthermore, a complex between the E4 ORF6 and ElB 55-kDa proteins promotes the selective nuclear export of late viral transcripts [107] and references therein).…”