Adenovirus early region 4 (E4) encodes proteins critical in a variety of processes required for a successful viral infection. While E4 is not required for DNA replication, three E4 proteins (E4 11k, product of E4 ORF3; E4 34k, product of E4 ORF6; and the product of E4 ORF4) regulate replication in infected cells. In addition, most of the viral DNA produced by E4 mutants lacking E4 11k and E4 34k is structurally abnormal, consisting of concatemers of the viral genome up to 6 or more monomers in length. Concatemeric viral DNA is not observed in cells infected by wild-type virus. Eukaryotic cells possess efficient double strand break repair (DSBR) systems for rejoining DNAs broken by radiation and other agents. To test the hypothesis that the concatenated viral DNA seen in E4 mutant infections arises by end-to-end joining of linear intracellular viral DNAs by DSBR, concatemer formation was examined in cells lacking the DNA-dependent protein kinase (DNA PK), an essential component of the cellular DSBR system. No concatemers were observed in E4 mutant infections of DNA PK- cells, consistent with the hypothesis that concatemers arise by DSBR and suggestion that in wild-type infections, E4 prevents concatenation by inhibiting DSBR. Further, E4 34k inhibited V(D)J recombination, a process that requires DSBR, in a plasmid-based assay. Finally, immunoprecipitation experiments showed that both the E4 11k and E4 34k proteins associate physically with DNA PK. Together, these data strongly suggest that both E4 proteins inhibit DSBR, possibly by a mechanism that involves binding to DNA PK. It is likely that suppression of concatemer formation increases the efficiency of viral DNA replication. Additionally, since DNA PK is a proximal element in the pathway that induces p53 activity in response to DNA, inhibition of DNA PK may be anti-apoptotic in infected cells. Thus, the interaction of E4 with DSBR may contribute in two distinct and novel ways to the success of an adenoviral infection. The goal of the work proposed here is to develop an understanding of the physical nature of the interactions between these E4 products and DNA PK, and to determine the significance to the viral life cycle of this newly-recognized aspects of E4 function.
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