During Adeno-Associated Virus and Adenovirus (AAV/Ad) coinfection, accumulation of viral genomes and proteins can alter cellular stress responses. To determine how AAV/Ad coinfection affects the host we screened over 60 cellular proteins for their responses. AAV/Ad coinfections induce a robust DNA damage response (DDR) that is distinct from that induced by Ad infection alone. Using chemical inhibitors, deficient cell lines and siRNA knockdowns of the DDR kinases, ATM, ATR and DNA-PK, we determined that DNA-PK and ATM kinases are the initial transducers of this response. AAV/Ad coinfection induces ATM-and DNA-PK mediated phosphorylation of RPA2, NBS1, H2AX and the checkpoint kinases CHK1/2. Inhibition of one or more of the DDR kinases reduces the level of phosphorylation of downstream targets but does not dramatically reduce Ad or AAV protein expression. However, AAV DNA levels are moderately affected by kinase inhibition. These experiments provide new insights into the cellular responses to AAV/Ad coinfections.

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