The E4orf6/E1B55K E3 Ubiquitin Ligase Complexes of Human Adenoviruses Exhibit Heterogeneity in Composition and Substrate Specificity

Abstract

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 ...

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

“…Previous work by our group and others has shown that a heterogeneity exists in Cullin binding among human adenovirus species (19,20). Whereas Ad5, representing species C, Ad34 (species B2), Ad9 (D), and Ad4 (E) bind essentially only Cul5, Ad12 (A) and Ad40 (F) bind essentially only Cul2.…”

“…Cul2 was knocked down in H1299 cells (19) by transfection with a plasmid DNA carrying short hairpin RNA (shRNA) against Cul2. The sequence for the shRNA was inserted between EcoRI and BglII sites of the pcDNA3 plasmid: 5=-A GCACCTGAACTGCTTGCT TTCAAGAGA AGCAAGCAGTTCAGG TGCT TTTTT-3=, with the hairpin listed in bold.…”

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

“…In HAdV-infected cells, E1B-55K and E4-34K form a complex with Cul-5 or Cul-2-containing E3 ubiquitin ligases to degrade cellular substrates such as p53. 4,5,9 However, this virus-commandeered E3 ligase may not play a major role in degrading Mdm2 and Mdm4, as both are efficiently degraded in cells infected with a dl1520 mutant virus that does not produce E1B-55K (Fig. 3).…”

“…5,6,9 Similarly, Cul5 NTD expression failed to stabilize Mdm2 and Mdm4 in Ad12-infected cells. In contrast, in Ad5-infected cells, Cul5 NTD expression resulted in reduced levels of Mdm2 (Fig.…”

“…For example, E1B-55K and E4orf6-34K proteins assemble an E3 ligase complex with cullin 2 or 5 (Cul2 or Cul5), Elongins B and C and RING box protein 1 or 2 (Rbx1 or 2). [3][4][5][6] A number of Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNAmediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection.…”