Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication

Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly found in other RCR biological systems. Two proteins, Rep and Rep', are essential for PCV DNA replication in mammalian cells. In this work, replicative intermediates of PCV-infected porcine kidney (PK15) cells or copy-release of PCV genomes from a head-to-tail tandem construct (without Rep') in Escherichia coli were examined. In PK15 cells, replicative intermediates consistent with complementary-strand replication which converts single-stranded circular genome to double-stranded supercoiled DNA and RCR which generates single-stranded plus strand progeny genome were observed. To a lesser extent, intermediates suggestive of recombination-dependent replication were also detected. In Escherichia coli, copy release of the single-stranded circular PCV genome with conversion to a supercoiled molecule by complementary-strand synthesis was observed. However, replicative intermediates indicative of RCR were not detected.