Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

Abstract

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.The linear, double-stranded adenovirus genome remains predominantly monomeric throughout a wild-type adenovirus infection. In contrast, most of the viral DNA present in cells infected by mutants that lack early region 4 (E4) is in the form of end-to-end concatemers containing two to five or more unit-length genomes (6,36,37). The viral genomes present in concatemers are joined in all orientations (head to tail, head to head, and tail to tail), and the joints between genomes typically lack a few to hundreds of nucleotides from each of the component molecules (37). Concatemer formation does not occur in mutant human cells that lack either DNA-dependent protein kinase (DNA-PK) (6) or DNA ligase IV (36), each of which is a central component of the nonhomologous endjoining (NHEJ) DNA repair system, and concatemers presumably are assembled from monomeric genomes by NHEJ. Concatemer formation is also blocked in cells that lack the host Mre11 or NBS1 protein, each of which is a member with the Rad50 protein of the MRN complex, which is central in DNA damage signal transduction in eukaryotes.Genome concatenation is redundantly antagonized by two virus-encoded systems in wild-type adenovirus infections: the presence of either the E4 11k (E4orf3) protein or the complex formed by the E4 34k (E4orf6) and E1b 55k proteins prevents concatemer formation (36, 37). These systems act by different mechanisms, but inhibition of concatenation by both is at least partly explained by inactivation of the Mre11 protein. E4 11k induces sequestration of Mre11 in aggresomes, inhibit...

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation

“…1). Previous work with Ad5 has shown that two viral proteins, E1B-55K and E4orf6, hijack a CUL5-based ubiquitin ligase complex to target p53, MRN, and LIG4 for degradation (12,14,22). In contrast, the data presented here show that Ad12 E1B-55K is not required for TOPBP1 degradation (Fig.…”

“…1A). DNA ligase IV (LIG4), previously shown to be targeted for degradation in Ad5-infected cells (22), was also degraded in Ad12-infected cells, indicating that, like MRN, it may be a common target for human Ads. Further analysis revealed that ATR, ATRIP, RPA70, RPA32, and RAD9 levels remained constant in Ad5-and Ad12-infected cells.…”

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…This possibility is nevertheless fascinating as it would offer a possible explanation for the different transformation capabilities of the large adenovirus E1Bs and earlier suggestions linking nuclear localization of HAdV5 E1B-55K (Endter et al, 2005) and presumably of subgroup HAdV12 E1B-54K (Kra¨tzer et al, 2000) to their oncogenic potential. However, it remains subject for further studies to evaluate how and whether E1B-55K facilitates PML and/or associated proteins to mediate its multiple functions during productive infection involving transcriptional repression of p53 (Yew et al, 1994;Berk, 1998, 1999), selective protein degradation and viral mRNA transport in combination with E4orf6 Querido et al, 2001;Stracker et al, 2002;Baker et al, 2007;Dallaire et al, 2009).…”

Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies

“…The Ad5 E1B-55K⅐E4-ORF6 complex functions as an adaptor molecule in an E3 ubiquitin ligase complex (22,23). E1B-55K⅐E4-ORF6 target the MRN proteins, as well as DNA ligase IV and Blm helicase, for inactivation via ubiquitin-mediated, proteasome-dependent degradation (18,24,25). The E4-ORF3 protein facilitates this process by promoting the transport of the MRN proteins to cytoplasmic aggresomes (26,27).…”