Stracker Travis H, Cassell Geoffrey D, Ward Peter, Loo Yueh-Ming, van Breukelen Bas, Carrington-Lawrence Stacy D, Hamatake Robert K, van der Vliet Peter C, Weller Sandra K, Melendy Thomas, Weitzman Matthew D
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
J Virol. 2004 Jan;78(1):441-53. doi: 10.1128/jvi.78.1.441-453.2004.
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.
2型腺相关病毒（AAV）是一种人类细小病毒，其复制依赖于细胞蛋白以及辅助病毒提供的功能。在细胞培养中支持AAV复制的最小单纯疱疹病毒1型（HSV-1）蛋白是由UL5、UL8和UL52组成的解旋酶-引发酶复合物，以及UL29基因产物ICP8。我们发现AAV和HSV-1复制蛋白在离散的核内位点共定位。用突变基因转染表明解旋酶-引发酶的酶促功能并非必不可少。单独的ICP8蛋白在体外试验中增强了AAV的复制。我们还展示了在各种支持复制的条件下，细胞复制蛋白A（RPA）在AAV中心的定位。体外试验表明，AAV Rep68和Rep78蛋白与腺病毒（Ad-DBP）、HSV-1（ICP8）和细胞（RPA）的单链DNA结合蛋白（ssDBP）相互作用，并且这些蛋白增强了Rep蛋白在起始位点的结合和切口。这些结果突出了核内定位的重要性，并表明Rep与多种ssDBP的相互作用使AAV能够在多种条件下复制。
