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A proteomic approach to identify candidate substrates of human adenovirus E4orf6-E1B55K and other viral cullin-based E3 ubiquitin ligases.

ABSTRACT: It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin alpha3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.

SUBMITTER: Dallaire F

PROVIDER: S-EPMC2786712 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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A proteomic approach to identify candidate substrates of human adenovirus E4orf6-E1B55K and other viral cullin-based E3 ubiquitin ligases.

Dallaire Frédéric F Blanchette Paola P Branton Philip E PE

Journal of virology 20090916 23

It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have ...[more]

PMID: 19759146

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Project description: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.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.

Project description:Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication by enhancing the action of E1A products.

Dataset Information

A proteomic approach to identify candidate substrates of human adenovirus E4orf6-E1B55K and other viral cullin-based E3 ubiquitin ligases.

Publications

A proteomic approach to identify candidate substrates of human adenovirus E4orf6-E1B55K and other viral cullin-based E3 ubiquitin ligases.

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