The 11-kDa product of adenovirus early region 4 (E4) open reading frame (ORF) 3 participates in many processes occurring in infected cell, including post-transcriptional steps in late viral gene expression and viral DNA synthesis. In addition, E4ORF3 from adenovirus type 5 (Ad5) displays the features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 El genes and confers multiple additional transformed properties on E1-expressing cells. Biochemical details of E4ORF3 activities in these processes are not well understood. A large body of evidence indicates that its lytic and transforming functions are mediated by physical interactions with viral and cellular components involved in DNA transcription and repair, as well as by host cell factors that regulate the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). In this study we have employed the two-hybrid screen in yeast to isolate human cDNAs encoding for E4ORF3-interacting proteins. Among 15 positive clones five cDNAs encode for a cellular protein called AUP1. In vitro-binding assays demonstrated that AUP1 fused to glutathione S-transferase (GST) specifically binds to E4ORF3 from Ad5, Ad9 and Ad40 generated in a coupled transcription-translation system, whereas no interactions was observed with ORF3 from Ad12. Interestingly, GST-AUP1 interacted also specifically with in vitro translated Ad5 E1A proteins. Regions involved in the Ad5 E4ORF3/AUP1 interaction in vitro map to the central part of E4 protein and the carboxy-terminal region of AUP1, while E1A binds to an amino-terminal segment of the cell protein. Taken together, these studies indicate that AUP1 may represent a cellular target of both adenovirus E4ORF3 and E1A proteins. Additional studies are currently under way to confirm the significance of these interactions in living cells in vivo.