Adeno-Associated Virus 2 (AAV2) - Induced RPA exhaustion generates cellular DNA damage and restricts viral gene expression

Parvoviruses are single-stranded DNA viruses that have been modified to serve as vehicles for therapeutic transgene delivery in the form of recombinant Adeno-Associated Virus (rAAV2) vectors or rodent parvovirus-derived oncolytic agents. Infection with viruses of the Parvoviridae family induces a cellular DNA Damage Response (DDR) signal that supports virus replication. However, it remains unknown whether rAAV2 vectors or non-replicative wild-type AAV2 (wtAAV2) genomes induce cellular DDR signals, which might be deleterious to the cell. To determine the impact of wtAAV2/rAAV2 genomes on the integrity of the host chromosome, we have pulsed wtAAV2/rAAV2 infected cells with BrdU analogs followed by single-molecule imaging of the cellular replisomes and proteomic analysis of the host replication forks. We discovered that non-replicative wtAAV2/rAAV2 genomes are sufficient to induce replication stress on the host genome, leading to DDR signals in a dose-dependent manner. Moreover, infection with replication-competent wtAAV2 leads to enrichment of replication stress proteins, DNA repair factors and RNA processing machinery on cellular replication forks. However, neither the wtAAV2 Inverted Terminal Repeats (ITRs) that are retained in rAAV2s nor empty capsids are sufficient to induce host-cell replication stress. Strikingly, incoming wtAAV2 genomes associate with the single-stranded DNA binding protein RPA in host cells in a dose-dependent manner, progressively shortening cellular replication forks. These elevated levels of wtAAV2-induced cellular replication stress eventually leads to accumulation of DDR signals in the nucleus. Chemical inhibition of RPA activity and RNAi-mediated knockdown leads to de-repression of the wtAAV2 genome, increasing Rep 68/78 gene expression. Ectopic expression of RPA rescues wtAAV2-induced replication stress. Taken together, our findings suggest that depletion of cellular stores of RPA molecules by competing wtAAV2 genomes restrict viral gene expression and cause cellular DNA damage.