Adeno-associated virus (AAV) site-specific integration: Formation of AAV-AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of a deno-associated virus (AAV) was developed. This system is based on two subs trates. a linear or circular AAV donor and a circular acceptor containing t he preintegration locus AAVS1, In the presence of HeLa extract and the His- Tag-purified Rep68 protein, specific covalent junctions between AAV and AAV S1 were formed and detected by PCR. The majority of the junctions were loca ted within the Rep binding site of both the AAV and the AAVS1 substrates, u nderlining the involvement of the Rep protein. A limited amount of replicat ion and the presence of nuclear factors promoted the efficiency of the reac tion. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to cur rent models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integ ration of AAV into human chromosome 19 in vivo.