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Journal of Virology, March 2000, p. 2372-2382, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay

Toni Cathomen, Delphine Collete,dagger and Matthew D. Weitzman*

Laboratory of Genetics, The Salk Institute for Biological Studies, San Diego, California 92186

Received 13 October 1999/Accepted 7 December 1999

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

* Corresponding author. Mailing address: Laboratory of Genetics, The Salk Institute, P.O. Box 85800, San Diego, CA 92186. Phone: (858) 453-4100, ext. 2037. Fax: (858) 558-7454. E-mail: weitzman{at}salk.edu.

dagger Present address: Center of Molecular and Cellular Biology, Faculty of Agronomy, B-5030 Gembloux, Belgium.

Journal of Virology, March 2000, p. 2372-2382, Vol. 74, No. 5
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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