The primary focus of this project will be the targeting of DNA integration as mediated by the Rep proteins of adeno-associated virus (AAV). As shown previously, AAV integrates its genome site-specifically into human chromosome 19. Using an episome-based assay, we were able to demonstrate that a 33 nucleotide signal, present within the target sequence, is necessary and sufficient to mediate this highly specific integration/recombination event. The DNA targeting signal contains two sequence motifs which are also present in the viral origin of replication and which are bound by the viral Rep proteins. The hypothesis is that the viral Rep proteins determine site-specific integration through recognition and binding of these origin signals present on chromosome 19. This project will test the feasibility of retargeting Rep-mediated site-specific DNA integration by means of exchanging the DNA binding domain of the AAV Rep proteins. In order to establish a model system, we first propose to exchange the DNA binding domain of AAV Rep78 with its counterpart from goose parvovirus (GPV). Interestingly, the similarity between the two proteins exceeds 90 percent within the active domain of Rep. The DNA-binding domains share much lower similarity consistent with the observation that GPV Rep1 recognizes a different DNA motif. Following biochemical characterization of the hybrid protein a recombinant AAV will be generated containing a rep gene that encodes the hybrid protein. This virus will be used in an episome based integration assay to test for altered site-specific DNA integration into the sequence motif that is recognized by the GPV Rep1 protein. The second goal will be to redirect integration to sites within the human genome other than AAVS1. A genetic screen will be used to identify mutants of the Rep protein capable of binding defined cellular target sequences with high affinity. Once these mutant proteins are purified and biochemically characterized, the episomal integration assay will be used to test for integration into the new target sequences. The experiments proposed are designed to provide further insights into the mechanism of targeted DNA integration with regard to functions of AAV Rep as well as to provide us with a tool to target selected sites within the human genome.
| Ward, P; Falkenberg, M; Elias, P et al. (2001) Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system. J Virol 75:10250-8 |
| Nony, P; Tessier, J; Chadeuf, G et al. (2001) Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences. J Virol 75:9991-4 |
| Yoon, M; Smith, D H; Ward, P et al. (2001) Amino-terminal domain exchange redirects origin-specific interactions of adeno-associated virus rep78 in vitro. J Virol 75:3230-9 |
| Dutheil, N; Shi, F; Dupressoir, T et al. (2000) Adeno-associated virus site-specifically integrates into a muscle-specific DNA region. Proc Natl Acad Sci U S A 97:4862-6 |
| Ward, P; Linden, R M (2000) A role for single-stranded templates in cell-free adeno-associated virus DNA replication. J Virol 74:744-54 |
