The chicken anemia virus-derived protein Apoptin induces apoptosis specifically in human tumor and transformed cells and not in normal, untransformed cells. The cell killing activity correlates with a predominantly nuclear localization of Apoptin in tumor cells, whereas in normal cells, it is detected mainly in cytoplasmic structures. To explore the role of nuclear localization for Apoptin-induced cell death in tumor cells, we employed a mutagenesis strategy. First, we demonstrated that the C terminus of Apoptin contains a bipartite-type nuclear localization signal. Strikingly, further investigation showed that Apoptin contains two different domains that induce apoptosis independently, and for both domains, we found a strong correlation between localization and killing activity. Using inhibitors, we ruled out the involvement of de novo gene transcription and translation and further showed that Apoptin itself does not have any significant transcriptional repression activity, suggesting that Apoptin exerts its effects in the nucleus by some other method. To determine whether nuclear localization is sufficient to enable Apoptin to kill normal, untransformed cells, we expressed fulllength Apoptin fused to a heterologous nuclear localization signal in these cells. However, despite its nuclear localization, no apoptosis was induced, which suggests that nuclear localization per se is not sufficient for Apoptin to become active. These studies increase our understanding of the molecular pathway of Apoptin and may also shed light on the mechanism of cellular transformation.

Apoptin, a protein from chicken anemia virus without an apparent cellular homologue, can induce apoptosis in mammalian cells. Its cytotoxicity is limited to transformed or tumor cells, making Apoptin a highly interesting candidate for cancer therapy. To elucidate Apoptin's mechanism of action, we have searched for binding partners in the human proteome. Here, we report that Apoptin interacts with DEDAF, a protein previously found to associate with death effector domain (DED)-containing pro-apoptotic proteins, and to be involved in regulation of transcription. Like Apoptin, after transient overexpression, DEDAF induced apoptosis in various human tumor cell lines, but not in primary fibroblasts or mesenchymal cells. DEDAF-induced cell death was inhibited by the caspase inhibitor p35. Together with the reported association of DEDAF with a DED-containing DNA-binding protein in the nucleus and the transcription regulatory activity, our findings may provide a clue for the mechanism of Apoptin's actions in mammalian cells.