Characterization of the Rep78/Adeno-Associated Virus Complex

Abstract

Adeno-associated virus (AAV) replication proteins Rep78 and Rep68 play major roles in the life cycle of AAV. We have recently providedin vivoevidence for the existence of a covalent association between Rep78 and virion single-stranded (ss) AAV DNA (K. M.R. Prasad and J.P. Trempe(1995)Virology214, 360–370). In this work we have further characterized the Rep78 protein–AAV DNA covalent linkage. Exonuclease and primer extension analyses revealed that in the majority of isolated ssDNA, Rep78 protein is covalently linked to one of the 5′ terminal thymidines. Pulse–chase experiments with radiolabeled methionine suggest that Rep protein remains associated with the virus particle for up to 8 hr after labeling in infected cells. Quantitative immunoprecipitation indicated that ∼30% of the ssDNA remains associated with the Rep protein after cell fractionation and partial purification. When cells are infected with Rep-associated AAV particles, a significant proportion of viral DNA remains attached to Rep after entry into the nucleus. However, the linkage does not persist after nuclear entry. These observations suggest that covalently linked Rep78 protein may play a key role during wild-type AAV infections and AAV vector transductions.