title: The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication.
abstract: 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.
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors.forename: Travis H
authors.initials: TH
authors.lastname: Stracker
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Cassell
authors.forename: Peter
authors.initials: P
authors.lastname: Ward
authors.forename: Yueh-Ming
authors.initials: YM
authors.lastname: Loo
authors.forename: Bas
authors.initials: B
authors.lastname: van Breukelen
authors.forename: Stacy D
authors.initials: SD
authors.lastname: Carrington-Lawrence
authors.forename: Robert K
authors.initials: RK
authors.lastname: Hamatake
authors.forename: Peter C
authors.initials: PC
authors.lastname: van der Vliet
authors.forename: Sandra K
authors.initials: SK
authors.lastname: Weller
authors.forename: Thomas
authors.initials: T
authors.lastname: Melendy
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Weitzman
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology; D006801:Humans; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2