Impact of DNA damage proteins on the adenoviral lifecycle

Viruses manipulate the cellular environment to promote productive infection. Cells mount anti-viral defenses to combat and limit viral productivity. Viruses in turn either hijack the anti-viral responses or inactivate them in order to overcome cellular defenses. Adenovirus (Ad) has been shown to inactivate the cellular DNA damage response by targeting the sensor for DNA damage, the MRN complex (composed of Mre11, Rad50, and Nbs1). Our lab has shown that the adenoviral E1B55K/E4orf6 complex and E4orf3 proteins inactivate MRN. E1B55K/E4orf6 promote the degradation of the MRN complex, while E4orf3 mislocalizes MRN into intranuclear tracks. Infection with adenovirus lacking the E4 region is less productive than wild type Ad5 infection and fails to inactivate MRN, which results in activation of the DNA damage response and concatemerization of viral genomes. The impact of the DNA damage response proteins on the viral lifecycle was not well understood. The work presented in this thesis provides evidence that the DNA damage response proteins negatively affect the adenoviral lifecycle at multiple stages. Using mutants of E1B55K, we found that degradation of MRN requires distinct domains of E1B55K and promotes viral late protein synthesis. We demonstrated that production of viral late proteins occurs independently of concatemer formation. Using hypomorphic cell lines we have found that the MRN complex negatively impacts viral DNA replication independently of both concatemer formation and signaling by DNA damage response kinases, ATM and ATR. We concluded that the viral proteins E1B55K, E4orf6, and E4orf3, promote viral DNA replication by inactivating the MRN complex. Additionally, we found that ATR signaling, but not ATM signaling, negatively impacts the accumulation of late viral mRNA during [Delta]viral infection. We believe that the DNA damage response has multiple ways to limit adenoviral infection and that the virus has evolved strategies to inactivate these responses. Our data elucidate the anti-viral activity of the MRN complex during adenoviral infection, and provides further insight into the functions of MRN and ATR signalin