Induction of transformation and p53-dependent apoptosis by adenovirus type5 E4orf6/7 cDNA

Adenovirus (Ad) E4orf6/7, one of the early gene products of human Ads, form s a stable complex with the cellular transcription factor E2F to activate t ranscription from the Ad E2 promoter. E2F cDNAs have growth-promoting and a poptosis-inducing activities when overexpressed in cells. We cloned Ad5 E40 rf6/7 cDNA in both simian virus 40- and human cytomegalovirus-based express ion vectors to examine its transforming and apoptotic activities. The clone d E4orf6/7 collaborated with a retinoblastoma protein (RB)-nonbinding and t herefore E2F-nonreleasing mutant of Ad5 ELA (dl922/947) to morphologically transform primary rat cells, suggesting that E2F is an important cellular p rotein functioning downstream of E1A for transformation. In a G418 colony f ormation assay, E4orf6/7 was shown to suppress growth of untransformed rat cells. Moreover, a recombinant Ad expressing Ad5 E4orf6/7 induced apoptosis in rat cells when coinfected with wild-type p53-expressing Ad. Mutational analysis of E4orf6/7 revealed that both of the domains required for growth inhibition and transformation by E4orf6/7 lay in the C-terminal region, whi ch is essential for transactivation from the upstream sequence of an E2a pr omoter containing E2F-binding sites. However, the smallest mutant of E4orf6 /7, encoding the C-terminal 59 amino acids, failed to complement the RE-non binding dl/922/947 mutant despite showing growth inhibition and E2F transac tivation activities. Thus, it is suggested that a subregion of E40rf6/7 whi ch is required for growth inhibition and transformation in collaboration wi th dl922/947 overlaps the transactivation domain of E4orf6/7.