Viral Modulation of Genetic Stability

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DESCRIPTION (provided by applicant): Viruses provide powerful tools to study fundamental cellular mechanisms. Using human adenovirus as a model we have identified the cellular DNA repair machinery as an obstacle to viral replication. Cellular DNA repair pathways produce concatemers of the double-stranded viral genome during infection with viruses deleted of the E4 region. The Mre11/Rad50/NBS1 complex is essential for concatemer formation. This cellular complex is important for double-strand DNA break repair, meiotic recombination and telomere maintenance. Defects in Mre11 and NBS1 genes predispose to malignancy in AT-like disorder (ATLD) and Nijmegen breakage syndrome respectively. We show that adenovirus possesses two mechanisms to inactivate this cellular complex. One viral protein (E4orf3) causes mislocalization of the Mre11 complex and two other viral proteins (E4orf6/E1b55K) induce degradation of Mre11 and Rad50 proteins. These viral proteins prevent concatemerization and also cause cell transformation using a "hit-and-run" strategy. We propose to investigate the role of the Mre11 complex in concatemer formation and the mechanism used by the virus to target this complex for degradation. We will use biochemical and molecular approaches to determine the activities of the Mre11 complex required for concatemerization of viral genomes. We will examine interactions of the Mre11 complex with the viral proteins and determine whether the E1b55K/E4orf6 complex can ubiquitinate the Mre11 protein to cause its degradation. We have observed that infection with viruses lacking the E4orf6/E1b55K complex results in activation of DNA repair signaling pathways. We will study these events and determine how they are blocked by E4orf6/E1b55K. We will also look at the consequences for the cell of Mre11 degradation and its effect on DNA repair. These studies will provide insights into cellular pathways that sense and respond to DNA damage and will also shed light on how viral oncoproteins cause transformation.