ROLE OF THE ADENOVIRUS DNA-BINDING PROTEIN IN IN-VITRO ADENOASSOCIATED VIRUS-DNA REPLICATION

A basic question in adeno-associated virus (AAV) biology has been whet her adenovirus (Ad) infection provided any function which directly pro moted replication of AAV DNA. Previously in vitro assays for AAV DNA r eplication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demons trated that Ad infection provides a direct helper effect for AAV DNA r eplication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of dire ct participation of Ad proteins in AAV DNA replication. In this report , we show that in the in vitro assay, enhancement of processivity occu rs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [ RPA]). Clearly Ad-DBP is present after Ad infection but not before, wh ereas the cellular level of RPA is not apparently affected by Ad infec tion. However, we have not measured possible modifications of RPA whic h might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmi d vector, RPA was not an effective substitute for Ad-DBP. Extracts sup plemented with Ad-DBP preferentially replicated AAV sequences rather t han adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.