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The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

Travis H. Stracker,^1,2, Geoffrey D. Cassell,¹ Peter Ward,³ Yueh-Ming Loo,⁴ Bas van Breukelen,⁵ Stacy D. Carrington-Lawrence,⁶ Robert K. Hamatake,^7, Peter C. van der Vliet,⁵ Sandra K. Weller,⁶ Thomas Melendy,⁴ and Matthew D. Weitzman^1*

Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037,¹ Graduate Program, Department of Biology, University of California, San Diego, California 92093,² Institute for Gene Therapy and Molecular Medicine, Mt. Sinai School of Medicine, New York, New York 10029,³ Departments of Microbiology and Biochemistry, School of Medicine and Biomedical Sciences, University of Buffalo, Buffalo, New York 14214,⁴ Department of Physiological Chemistry and Center for Biomedical Genetics, University Medical Center, 3584 GC Utrecht, The Netherlands,⁵ Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030,⁶ Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492⁷

Received 21 July 2003/ Accepted 18 September 2003

Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.

Present address: Memorial Sloan Kettering Cancer Center, New York, NY 10021.

Present address: Ribapharm, Inc., Costa Mesa, CA 92626.

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