The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion

Abstract

Resolution of the covalently closed terminus of adeno-associated Virus (AAV) DNA is mediated by viral replication protein Rep78 or Rep68. In vitro studies with purified Rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5' end of the viral genome. The in vivo existence and fate of the covalently associated Rep protein during the virus life cycle has not yet been elucidated. In this report, we use immunoprecipitation analyses to demonstrate that the Rep78 protein is covalently attached to viral DNA in a preformed virion. The attached Rep78 is susceptible to antibody binding and protease digestion, and the DNA linkage is susceptible to nuclease digestion, therefore Rep78 is probably located on the outside of the particle. Rep proteins are also attached to double-stranded replicative-form monomer (RFM) DNA in extracts from AAV and adenovirus coinfected cells. Rep protein attachment to RFM and encapsidated AAV DNA suggest that the covalent complex is an intermediate in virus assembly. These observations are similar to those noted by others for the autonomous parvoviruses and provide additional insights into parvovirus assembly.