UC San Diego

Viruses are highly evolved entities that target key cellular pathways in order to promote their own reproduction. However, host cells encode a number of defenses that obstruct productive infection, and viruses must either employ these to their advantage or disable them. A growing body of research is indicating that viruses must contend with cellular DNA damage response pathways that can affect virus replication. Adenovirus (Ad) is a DNA virus encoding proteins that defend against host DNA repair functions. Infection of cells with an E4 deleted Ad induces a cellular DNA damage response mediated by ATM and ATR, and mutant viral genome concatemerization. The adenoviral E1b55K/E4orf6 complex prevents these activities, and degrades a number of cellular factors, including p53, the Mre11 complex (MRN), and DNA Ligase IV. We examined the mechanism of E1b55K/E4orf6 mediated degradation of substrates and found that these events were separable. Intriguingly, analysis of cellular factors involved in down-regulation of targets suggested that E1b55K/E4orf6 might assemble distinct ubiquitin ligases. We also defined some of the viral requirements for degradation, and described a set of E1b55K mutants that can distinguish between substrates. Using these E1b55K separation-of-function mutants, we examined the consequences of substrate degradation on cellular responses. Through these experiments, we found that MRN is critical to mounting a DNA damage response to mutant Ad infection and exogenous DNA damage. While down-regulation of MRN can prevent mutant viral genome concatemerization, DNA Ligase IV degradation can also compensate. These E1b55K/E4orf6 activities had additional consequences for the small DNA virus, adeno-associated virus (AAV). Adenoviral proteins, including E1b55K/E4orf6, provide helper functions for AAV production. We found that MRN is a barrier to AAV replication, and its degradation is a critical helper activity of E1b55K/E4orf6. Interestingly, AAV and Ad co-infection induces a DNA damage response independent of MRN, which is degraded by Ad. Unlike an E4- deleted Ad infection, which triggers ATM and ATR, our data shows that a subset of signaling events during Ad and AAV co-infection are dependent on DNA-PK. Our experiments also indicate that DNA-PK has a role in AAV transduction and replication, and that this PIKK might be influenced by MRN. The work presented in this dissertation elucidates functions of the adenoviral E1b55K and E4orf6 proteins in both the adenovirus and AAV lifecycles. Our results also demonstrate how these viral proteins affect the cellular machinery for protein degradation and DNA repair. Our data prove that adenovirus and AAV provide powerful model systems to gain insight into these cellular pathways