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...

The adenovirus oncoproteins E4 34k and E4 11k, the products of E4 open reading frames 6 and 3, respectively, individually prevent the formation of concatemers of the linear viral genome in infected cells. We show here that genome concatenation in E4 mutant-infected cells requires the cellular DNA-dependent protein kinase (DNA PK) and that E4 34k inhibits V(D)J recombination, a normal cellular process that is also dependent on DNA PK. We further show that both E4 34k and E4 11k coimmunoprecipitate with DNA PK. These observations indicate that E4 products block formation of concatemers of the viral genome by inhibiting DNA PK-dependent double strand break repair and suggest that they act by forming a physical complex with DNA PK. DNA PK also participates in activation of p53 DNA-binding activity by DNA damage. By inhibiting DNA PK function, E4 products may block p53 activation in response to the products of viral DNA replication and thus provide a new mechanism to prevent apoptosis of infected cells.