Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin ␣3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin ␣3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.The human adenovirus type 5 (Ad5) early region 4 34-kDa product from open reading frame 6 (E4orf6) and the E1B55K protein have been known for some time to act in concert to carry out several important functions during the infectious cycle, including regulation of the activity and stability of p53 and, late in infection, the selective transport of viral mRNAs (2,14,18,19,37,46,51,52,56). Our group and others have shown that the cooperative functions of these Ad5 proteins, including the E4orf6-E1B55K interaction itself, appear to require formation of a Cul5-based E3 ubiquitin ligase complex (8). We showed that Ad5 E4orf6 recruits an E3 ubiquitin ligase complex containing the Cullin family member Cul5, Elongins B and C, and the RING protein Rbx1 (8,20). E1B55K appears to associate with the E4orf6 protein only in the context of this complex, and it is believed to function as the substrate recruitment component, introducing specific proteins for ubiquitination and degradation by proteasomes (8,11,28). The formation and function of this complex are essential to permit efficient viral replication. At one time p53 was its only known substrate (10,34,36,40,47,48); however, a growing list of additional targets is emerging, including the cellular proteins Mre11 (8, 49), DNA ligase IV (4), and ...

The E4orf6 protein of serotypes representing all human adenovirus species forms Cullin-based E3 ubiquitin ligase complexes that facilitate virus infection by inducing degradation of cellular proteins that impede efficient viral replication. This complex also includes the viral E1B55K product believed to bind and introduce substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as some serotypes form Cul5-based complexes whereas others utilize Cul2. Significant variations in substrate specificities also exist among serotypes, as some degrade certain substrates very efficiently whereas others induce more modest or little degradation. As E1B55K is believed to function as the substrate acquisition component of the ligase, we undertook studies to compare the ability of representative E1B55K proteins to bind substrates with the efficacy of degradation by their respective E4orf6-based ligases. Interestingly, although efficient degradation in some cases corresponded to the ability of E1B55K to bind to or relocalize substrates, there were several examples of substrates that bound efficiently to E1B55K but were not degraded and others in which substrates were degraded even though binding to E1B55K was low or undetectable. These results suggest that transient interactions with E1B55K may be sufficient for efficient substrate degradation and that binding alone is not sufficient, implying that the orientation of the substrate in the ligase complex is probably crucial. Nevertheless, we found that the substrate specificity of certain E4orf6-based ligases could be altered through the formation of hybrid complexes containing E1B55K from another serotype, thus confirming identification of E1B55K as the substrate acquisition component of the complex.

bMuch of the work on the basic molecular biology of human adenoviruses has been carried out on a very limited number of the more than 60 serotypes, primarily the highly related species C viruses adenovirus type 5 (Ad5) and Ad2 and, to some extent, Ad12 of species A. Until recently, it has been widely assumed that insights obtained with these model viruses were representative of all human adenoviruses. Recent studies on the E3 ubiquitin ligase formed by the viral E1B55K and E4orf6 proteins with a cellular Cullin-based complex indicated that although all species form such a functional complex, significant variations exist in terms of complex composition and the substrates that are degraded. In the present report we conducted a comprehensive analysis of the localization of E1B55K products from representatives of six of the seven adenovirus species in the presence and the absence of the corresponding E4orf6 protein. We found that although in some species E1B55K localized in aggresomes, such was not always the case, suggesting that these structures are not necessary for the efficient degradation of substrates. In addition, differences were evident in the localization of E1B55K, although all forms readily associated with PML. Finally, Ad5 E1B55K was seen to localize in close proximity to Rab11, a marker for the endosomal recycling compartment, and both focused at the microtubule organizing center. These findings suggest that E1B55K from some species may employ the transport system utilized by the membrane recycling pathway to assemble aggresomes and the possibility that this structure might then affect recycling of cell surface components.

The human adenovirus E4orf6 and E1B55K proteins are part of an E3 ubiquitin ligase complex that degrades p53, Mre11 and probably other cellular polypeptides. Our group has demonstrated previously that this complex contains Cul5, Rbx1 and Elongin B and C and is formed through interactions of these cellular proteins with E4orf6. Although this E4orf6 complex is similar in many ways to the cellular SCF and VBC E3 ligase complexes, our previous work indicated that unlike all known Cullin-containing complexes, E4orf6 contains two functional BC-box motifs that permit interactions with Elongin B and C. Here we show that a third BC-box exists that also appears to be fully functional. In addition, we attempted to identify a region in E4orf6 responsible for the specific selection of Cul5, which we show herein by knocking down Cul5 protein levels, is essential for p53 degradation. One sequence within E4orf6 shares limited homology with the 'Cul5 box motif', a recently identified sequence found to be responsible for selection of Cul5 in some cellular Cullin-containing E3 ligase complexes; however, genetic analysis indicated that this motif is not involved in Cullin binding or p53 degradation. Thus E4orf6 appears to utilize a different mechanism for Cul5 selection, and, both in terms of interactions with Elongin B and C and with Cul5, assembles the E3 ligase complex in a highly novel fashion.

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. IMPORTANCEThe 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.