Demonstration of Nicking/Joining Activity at the Origin of DNA Replication Associated with the Rep and Rep′ Proteins of Porcine Circovirus Type 1

Abstract

The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3'-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep' have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep' is as yet unknown. In this study, the ability of PCV1 Rep and Rep' to "nick" and "join" strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5'-TAGTATTAC-3') located at the apex of a putative stem-loop structure. In addition, the Rep and Rep' proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual "nicking/joining" activities associated with PCV1 Rep and Rep' are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.