The adeno-associated virus 2 genome and Rep 68/78 proteins interact with cellular sites of DNA damage

Nuclear DNA viruses simultaneously access cellular factors that aid their life cycle while evading inhibitory factors by localizing to distinct nuclear sites. Adeno-associated viruses (AAVs), which are Dependoviruses in the family Parvovirinae, are non-enveloped icosahedral viruses, which have been developed as recombinant AAV vectors to express transgenes. AAV2 expression and replication occur in nuclear viral replication centers (VRCs), which relies on cellular replication machinery as well as coinfection by helper viruses such as adenoviruses or herpesviruses, or exogenous DNA damage to host cells. AAV2 infection induces a complex cellular DNA damage response (DDR), in response to either viral DNA or viral proteins expressed in the host nucleus during infection, where VRCs co-localized with DDR proteins. We have previously developed a modified iteration of a viral chromosome conformation capture (V3C-seq) assay to show that the autonomous parvovirus minute virus of mice localizes to cellular sites of DNA damage to establish and amplify its replication. Similar V3C-seq assays to map AAV2 show that the AAV2 genome co-localized with cellular sites of DNA damage under both non-replicating and replicating conditions. The AAV2 non-structural protein Rep 68/78, also localized to cellular DDR sites during both non-replicating and replicating infections, and also when ectopically expressed. Ectopically expressed Rep could be efficiently re-localized to DDR sites induced by micro-irradiation. Recombinant AAV2 gene therapy vector genomes derived from AAV2 localized to sites of cellular DNA damage to a lesser degree, suggesting that the inverted terminal repeat origins of replication were insufficient for targeting.