A Functional Complex of Adenovirus Proteins E1B-55kDa and E4orf6 Is Necessary To Modulate the Expression Level of p53 but Not Its Transcriptional Activity

Abstract

In adenovirus-infected cells, binding of E1B-55kDa and E4orf6 to the tumor suppressor protein p53 inhibits its transcriptional activity and causes rapid turnover of the protein. To investigate the requirements of the E1B-E4orf6 complex to modulate p53 function, we generated an E4orf6 mutant that failed to associate functionally and physically with E1B-55kDa but still interacted with p53. We confirm that E4orf6 and E1B-55kDa reduce p53 transactivation individually and show that their combined inhibition is additive rather than synergistic. Furthermore, we found that downregulation of p53's expression level, but not transcriptional inhibition of p53, depends on a functional E1B-E4 complex. A functional interaction of E1B-55kDa with p53, on the other hand, is a prerequisite for both transcriptional repression and downregulation of p53. The separation of these two functions will enable further dissection of the requirements for oncogenicity by the E4orf6 protein.

Adenoviral E1B55K oncoprotein sequesters candidate leukemia suppressor sequence-specific single-stranded DNA-binding protein 2 into aggresomes

“…Finally, coexpression of the adenoviral E4orf6 protein results in nuclear accumulation of E1B55K (Ornelles and Shenk, 1991;Cathomen and Weitzman, 2000). When E4orf6 is expressed in 293 cells, SSBP2 is found to be colocalized with E1B55K in the nucleus and the aggresome is no longer apparent (Figure 4d).…”

“…Simultaneously, E1B55K inactivates the p53 pathway by inhibiting p53-mediated transcription, thereby preventing growth arrest and apoptosis (Yew and Berk, 1992;Yew et al, 1994;Teodoro and Branton, 1997;Martin and Berk, 1998). During adenovirus infection, E1B55K also associates with the viral E4orf6 protein and cellular proteins, forming an E3 ubiquitin ligase complex that induces proteasomal degradation of p53 (Querido et al, 1997(Querido et al, , 2001aSteegenga et al, 1998Steegenga et al, , 1999Cathomen and Weitzman, 2000;Wienzek et al, 2000;Harada et al, 2002). The E1B55K/E4orf6 complex also degrades cellular DNA repair proteins to prevent a DNA damage response (Stracker et al, 2002;Carson et al, 2003) and is required for RNA transport and late protein synthesis during infection (Dobner and Kzhyshkowska, 2001;Flint and Gonzalez, 2003).…”

Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: A DNA microarray analysis

“…Because the E4 gene of adenovirus has been implicated in affecting the cell cycle (37) and inactivating cellular p53 protein (38)(39)(40)(41), we determined whether the IE86 protein activated transcription of the adenovirus E4 gene. Similar levels of E4 expression were detected with the control Ad-GFP or with Ad-IE86 in transduced HFF cells.…”

Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

“…Immunofluorescence was basically performed as described 39,40 Cells were fixed after 30 h and stained with antibodies against γ-H2AX (top and right hand panels), HA (to detect ZFN) and DAPI, as indicated. Due to the lack of a nuclear localization signal, the ZFNs are localized throughout the cell.…”