Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfe ction with a helper virus, e.g., adenovirus or herpesviruses. In the case o f adenovirus coinfection, the replication machinery of the host cell perfor ms AAV DNA replication. In contrast, it has been proposed that the herpesvi rus replication machinery might replicate AAV DNA. To investigate this ques tion, 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 RSV-1 UL5, UL8, UL29, UL-30, UL42, and UL52 gene products along wi th the AAV Rep68 protein are sufficient to initiate replication on duplex D NA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates cont aining only a Rep binding site and a terminal resolution site. We further d emonstrate 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 pr otein also acts as a helicase for DNA synthesis. The single-strand DNA bind ing protein (the UL29 gene product) presumably prevents reannealing of comp lementary strands. These results suggest that AAV can use the HSV replicati on apparatus to replicate its DNA. In addition, they may provide a first st ep for the development of a fully reconstituted AAV replication assay.