Analysis of the Cullin binding sites of the E4orf6 proteins of human adenovirus E3 ubiquitin ligases.

UNLABELLED: E4orf6 proteins of human adenoviruses form Cullin-based E3 ubiquitin ligase complexes that degrade cellular proteins, which impedes efficient viral replication. These complexes also include the viral E1B55K product, which is believed to recruit most substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as serotypes representing some species form Cul5-based complexes (species B2, C, D, and E), whereas others utilize Cul2 (species A and F). Adenovirus type 16 (Ad16; species B1) binds significant levels of both. In this report, we show that the Cul2 binding sequence in E4orf6 of Ad12 (species A) and Ad40 (species F) resembles the cellular consensus Cul2 box. Mutation within this Cul2 box prevents binding not only of Cul2 but also in some cases Elongin C and reduces the ability to degrade target proteins, such as Mre11 and p53. A comparable Cul2 box is not present in E4orf6 of Ad5 and other serotypes that bind Cul5; however, creation of this Cul2 box sequence in Ad5 E4orf6 promoted binding to Cul2 and Cul2-dependent degradation of Mre11. E4orf6 of Ad16 also binds Cul2; however, unlike Ad40, it does not contain an Ad12-like Cul2 box, suggesting that Ad16 binds Cul2 in a unique but perhaps nonfunctional manner, as only Cul5 binding complexes appeared able to degrade Mre11. Our extensive analyses have thus far failed to identify a consensus Cul5 binding sequence, suggesting that association occurs via a novel and perhaps complex pattern of protein-protein interactions. Nevertheless, the identification of the Cul2 box may allow prediction of Cullin specificity for all E4orf6-containing Adenoviridae. IMPORTANCE: The work described in this paper is a continuation of our in-depth studies on the Cullin-based E3 ligase complexes formed by the viral E4orf6 and E1B55K proteins of all human adenoviruses. This complex induces the degradation of a growing series of cellular proteins that impede efficient viral replication. Some human adenovirus species utilize Cul5, whereas others bind Cul2. In this paper, we are the first to identify the E4orf6 Cul2 binding site, which conforms in sequence to a classic cellular Cul2 box. Ours is the first detailed biochemical and genetic analysis of a Cul2-based adenovirus ligase and provides insights into both the cooperative interactions in forming Cullin-based ligases as well as the universality of formation of all adenovirus ligase complexes. Our work now permits future analysis of the evolutionary significance of the ligase complex, work that is currently in progress in our lab.

Identification of the adenovirus E4orf4 protein binding site on the B55α and Cdc55 regulatory subunits of PP2A: Implications for PP2A function, tumor cell killing and viral replication.

Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) is required, and such binding inhibits PP2A(B55) activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2A(B55α). We propose that E4orf4 inhibits PP2A(B55) activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2A(B55) to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product.