Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylati on. Here we show that in transient transfection experiments, adenovirus EIA activation or the viral E2 promoter is abrogated by coexpression of the vi ral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to requ ire the retinoblastoma protein that previously has been shown to regulate E 2F activity. The inhibitory activity of E4-ORF4 appears to be specific beca use E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the 52 p romoter. In agreement with this, we find that E4-ORF4 inhibits E2F1/DP-1-me diated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expressi on during virus growth. E4-ORF4 has previously been shown to bind to and ac tivate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF 4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activ ity, suggesting that E4-ORF4 represses E2 transcription by inducing transcr iption factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 ex pression appears to result in reduced stability of E2F/DNA complexes. (C) P ress Academic Press.