Roles of Adeno-Associated Virus Rep Protein and Human Chromosome 19 in Site-Specific Recombination

ABSTRACT Adeno-associated virus type 2 (AAV) is the only known eucaryotic virus capable of targeted integration in human cells. AAV integrates preferentially into human chromosome (ch) 19q13.3qter. The nonstructural proteins of AAV-2, Rep78 and Rep68, are essential for targeted integration. Rep78 and Rep68 are multifunctional proteins with diverse biochemical activities, including site-specific binding to AAV and ch-19 target sequences, helicase activity, and strand-specific, site-specific endonuclease activities. Both a Rep DNA binding element (RBE) and a nicking site essential for AAV replication present within the viral terminal repeats are also located on ch-19. Recently, identical RBE sequences have been identified at other locations in the human genome. This fact raises numerous questions concerning AAV targeted integration; specifically, how many RBE sequences are in the human genome? How does Rep discriminate between these and the ch-19 RBE sequence? Does Rep interact with all sites and, if so, how is targeted integration within a fixed time frame facilitated? To better characterize the role of Rep in targeted integration, we established a Rep-dependent filter DNA binding assay using a highly purified Rep-68 fusion protein. Electron microscopy (EM) analysis was also performed to determine the characteristics of the Rep-RBE interaction. Our results determined that the Rep affinity for ch-19 is not distinct compared to other RBEs in the human genome when utilizing naked DNA. In fact, a minimum-binding site (GAGYGAGC) efficiently associated with Rep, suggesting that as many as 2 × 105 sites may exist. In addition, such sites also exist frequently in nonprimate mammalian genomes, although AAV integrates site specifically into primate genomes. EM analysis demonstrated that only one Rep-DNA complex was formed on ch-19 target DNA. Surprisingly, identically sized complexes were observed on all substrates containing a RBE sequence, but never on DNA lacking an RBE. Rep-DNA complexes involved a multimeric protein structure that spanned ca. 60 bp. Immunoprecipitation of AAV latently infected cells determined that 1,000 to 4,000 copies of Rep78 and Rep68 protein are expressed per cell. Comparison of the Rep association constant with those of established DNA binding proteins indicates that sufficient molecules of Rep are present to interact with all potential RBE sites. Moreover, Rep expression in the absence of AAVcis-acting substrate resulted in Rep-dependent amplification and rearrangement of the target sequence in ch-19. This result suggests that this locus is a hot spot for Rep-dependent recombination. Finally, we engineered mice to carry a single 2.7-kb human ch-19 insertion containing the AAV ch-19 target locus. Using cells derived from these mice, we demonstrated that this sequence was sufficient for site-specific recombination after infection with transducing vectors expressing Rep. This result indicates that any host factors required for targeting are conserved between human and mouse. Furthermore, the human ch-19 cis sequences and chromatin structure required for site-specific recombination are contained within this fragment. Overall, these results indicate that the specificity of targeted recombination to human ch-19 is not dictated by differential Rep affinities for RBE sites. Instead, specificity is likely dictated by human ch-19 sequences that serve as a Rep protein-mediated origin of replication, thus facilitating viral targeting through Rep-Rep interactions and host enzymes, resulting in site-specific recombination. Control of specificity is clearly dictated by the ch-19 sequences, since transfer of these sequences into the mouse genome are sufficient to achieve Rep-dependent site-specific integration.