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Journal of Virology, April 2001, p. 3230-3239, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3230-3239.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

Miran Yoon,1 Deborah H. Smith,1 Peter Ward,1 Francisco J. Medrano,2 Aneel K. Aggarwal,2 and R. Michael Linden1,3,*

Institute for Gene Therapy and Molecular Medicine,1 Department of Physiology and Biophysics,2 and Department of Microbiology,3 Mount Sinai School of Medicine, New York, New York 10029

Received 3 October 2000/Accepted 2 January 2001

The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame, REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

* Corresponding author. Mailing address: Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, 1 Gustave Levy Pl., Box 1496, New York, NY 10029-6574. Phone: (212) 659-8278. Fax: (212) 849-2437. E-mail: Michael.Linden{at}mssm.edu.

Journal of Virology, April 2001, p. 3230-3239, Vol. 75, No. 7
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.7.3230-3239.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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