CHARACTERIZATION OF THE REP78 ADENO-ASSOCIATED VIRUS COMPLEX/

Adeno-associated virus (AAV) replication proteins Rep78 and Rep68 play major roles in the life cycle of AAV. We have recently provided in vi vo evidence for the existence of a covalent association between Rep78 and virion single-stranded (ss) AAV DNA (K. M. R. Prasad and J. P. Tre mpe(1995) Virology 214, 360-370). In this work we have further charact erized the Rep78 protein-AAV DNA covalent linkage. Exonuclease and pri mer extension analyses revealed that in the majority of isolated ssDNA , Rep78 protein is covalently linked to one of the 5' terminal thymidi nes. 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 in dicated that similar to 0% of the ssDNA remains associated with the Re p protein after cell fractionation and partial purification. When cell s are infected with Rep-associated AAV particles, a significant propor tion of viral DNA remains attached to Rep after entry into the nucleus . However, the linkage does not persist after nuclear entry. These obs ervations suggest that covalently linked Rep78 protein may play a key role during wild-type AAV infections and AAV vector transductions. (C) 1997 Academic Press.