Analysis of the Cullin Binding Sites of the E4orf6 Proteins of Human Adenovirus E3 Ubiquitin Ligases

Abstract

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.

Cullin E3 Ligases and Their Rewiring by Viral Factors

“…This is exemplified in Adenoviruses, (viruses responsible for causing respiratory tract infections). Adenoviruses express E4ORF6 proteins that can bind both CRL2 and CRL5 host complexes in a serotype dependent fashion [ 26 ]. A common function across all serotypes is the ability of E4ORF6 proteins to form part of a functional CRL complex resulting in the degradation of host factors [ 100 ].…”

“…Preventing the apoptotic-inducing ability of p53 however has the unwanted effect of potentially causing extensive DNA damage within infected cells. Adenoviruses (like Ad5) circumvent this by additionally targeting the MRE11 subunit of the host DNA damage sensing MRN complex (consisting of MRE11A-RAD50-NBS1) for degradation using the same E4ORF6–EB155K–CRL5 [ 26 ]. As mentioned, not all serotypes of adenoviruses target the same substrates, E4ORF6 from Ad12 dampens host DNA damage signaling by degrading TOPBP1 rather than the MRN [ 30 , 101 ].…”

Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses

“…Such a motif was identified in HAdV-12 E4orf6 that matched the cellular protein consensus motif ⌽XXX⌽XXX⌽ (30,38). When this motif was altered through mutagenesis, E4orf6 lost the ability to bind Cul2 (38). Furthermore, when this motif was created in HAdV-5 E4orf6 at the equivalent position, binding to Cul2 was observed (38).…”

“…More success was obtained in our search for a Cul2 box. Such a motif was identified in HAdV-12 E4orf6 that matched the cellular protein consensus motif ⌽XXX⌽XXX⌽ (30,38). When this motif was altered through mutagenesis, E4orf6 lost the ability to bind Cul2 (38).…”

“…When this motif was altered through mutagenesis, E4orf6 lost the ability to bind Cul2 (38). Furthermore, when this motif was created in HAdV-5 E4orf6 at the equivalent position, binding to Cul2 was observed (38).…”