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Journal of Virology, November 2001, p. 10250-10258, Vol. 75, No. 21
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.21.10250-10258.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Rep-Dependent Initiation of Adeno-Associated Virus Type 2 DNA Replication by a Herpes Simplex Virus Type 1 Replication Complex in a Reconstituted System

Peter Ward,1,* Maria Falkenberg,2 Per Elias,2 Matthew Weitzman,3 and R. Michael Linden1,4

Institute for Gene Therapy and Molecular Medicine1 and Department of Microbiology,4 Mount Sinai School of Medicine, New York, New York; Department of Medical Biochemistry, Goteborg University, Goteborg, Sweden2; and Laboratory of Genetics, Salk Institute, San Diego, California3

Received 8 May 2001/Accepted 20 July 2001

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.

* Corresponding author. Mailing address: Institute for Gene Therapy and Molecular Medicine, Box 1496, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 659-8247. Fax: (212) 849-2437. E-mail: wardp01{at}doc.mssm.edu.

Journal of Virology, November 2001, p. 10250-10258, Vol. 75, No. 21
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.21.10250-10258.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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