Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat

Adeno-associated virus (AAV) is the only known eukaryotic virus capable of targeted integration in human cells. An AAV Rep binding element (RBE) and t erminal resolution site (trs) identical to the viral terminal repeats requi red for AAV DNA replication are located on chromosome (ch) 19. Both ch-19 R BE and trs elements have been shown to be essential for viral targeting to this locus. To characterize the role of the AAV inverted terminal repeat (I TR) cis-acting sequences in targeted integration an AAV trs mutant incapabl e of supporting viral replication was tested. Wild-type and mutant substrat es were assayed for targeted integration after cotransfection in the presen ce or absence of Rep. Our results demonstrated that, in the presence of Rep 78, both ITR substrates targeted to ch-19 with similar frequency. Molecular characterization of the mutant ITR integrants confirmed the presence of th e trs mutation in the majority of samples tested. Complementation analysis confirmed that the mutant targeted viral genomes were unable to rescue and replicate. In addition, Rep78 induced extensive rearrangement and amplifica tion of ch-19 sequences independent of wild-type or mutant targeting substr ate. These studies demonstrate that Rep-dependent nicking of the viral cis- acting trs sequence is not a prerequisite for site-specific recombination a nd suggests AAV targeting is mediated by Rep78/68-dependent replication fro m the ch-19 origin of replication (on). These studies have significant impa ct toward the understanding of AAV site-specific recombination and the deve lopment of targeting vectors.