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Journal of Virology, April 1999, p. 2930-2937, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Comparative Characterization of Rep Proteins from the Helper-Dependent Adeno-Associated Virus Type 2 and the Autonomous Goose Parvovirus

Deborah H. Smith,1 Peter Ward,2,dagger and R. Michael Linden1,*

Institute of Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, New York 10029,1 and Hearst Microbiology Research Center, Department of Microbiology, Cornell Medical College, New York, New York 100212

Received 9 October 1998/Accepted 4 January 1999

Adeno-associated viruses (AAVs) are nonautonomous human parvoviruses in that they are dependent on helper functions supplied by other viruses or on genotoxic stimuli for conditions permissive for replication. In the absence of helper, AAV type 2 enters latency by integration into a specific site on human chromosome 19. This feature of AAV, in combination with a lack of pathogenicity, makes AAV an attractive candidate vector for human gene therapy. Goose parvovirus (GPV) is both autonomous and pathogenic yet is highly homologous to AAV. To address the molecular bases for the different viral lifestyles, we compare the AAV and GPV nonstructural proteins, Rep78 and Rep1, respectively. We find that Rep78 and Rep1 possess several biochemical activities in common, including (i) high-affinity DNA binding for sequences that constitute the minimal DNA replication origin; (ii) nucleoside triphosphate-dependent DNA helicase activity; and (iii) origin-specific replication of double-stranded linear DNA. These experiments also establish a specific 38-bp DNA sequence as the minimal GPV DNA replication origin. It is noteworthy that although the proposed Rep binding sites of GPV and AAV are highly similar, Rep1 and Rep78 show a high degree of specificity for their respective origins, in both binding and replication assays. One significant difference was observed; with the minimal replication origin in adenovirus-uninfected extracts, Rep78-mediated replication exhibited low processivity, as previously reported. In contrast, Rep1 efficiently replicated full-length template. Overall, our studies indicate that GPV Rep1 and AAV Rep78 support a comparable mode of replication. Thus, a comparison of the two proteins provides a model system with which to determine the contribution of Rep in the regulation of dependence and autonomy at the level of DNA replication.

* Corresponding author. Mailing address: Institute of Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, 1 Gustave Levy Pl., Box 1496, New York, NY 10029. Phone: (212) 824-7740. Fax: (212) 849-2437. E-mail: lindem01{at}doc.mssm.edu.

dagger Present address: Institute of Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, NY 10029.

Journal of Virology, April 1999, p. 2930-2937, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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