Submitted to: European Society for Veterinary Virology
Publication Type: Abstract Only
Publication Acceptance Date: August 15, 2006
Publication Date: September 24, 2006
Citation: Cheung, A.K. 2006. Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli [abstract]. 7th International Congress of European Society for Veterinary Virology. Paper No. 049. Technical Abstract: 1. Introduction. Porcine circovirus (PCV) is a member of the Circoviridae family, which includes a group of diverse animal viruses with small, closed circular, single-stranded DNA that replicates their genome through double-stranded intermediates. Previous studies have shown that infectious virus can be generated from bacterial constructs containing head-to-tail tandem repeat PCV genomes with two origins of DNA replication (Oris) upon transfection into mammalian tissue culture cells or introduction into live animals. In this study, we examined the replication mechanisms of two greater than unit-length head-to-tail PCV1/PCV2 heterologous tandem constructs in Escherichia coli (E. coli). Heterologous tandem constructs were chosen because the components essential for PCV1 or PCV2 DNA replication (the Ori-IR and the Rep-complex) are interchangeable and the two viral genomes are different enough to be easily discernable. Furthermore, these constructs allowed easier identification of the mechanisms for generation of novel DNA species. 2. Material and methods. Two constructs, each consisting of 1.75 copies of PCV1/PCV2 DNA with two PCV Ori (specific for RCR mechanism) inserted into the pBluescript SK+ (pSK+) bacterial plasmid containing the colicin E1 (ColE1) Ori (specific for unidirectional theta-replication mechanism), were transformed into E. coli. 3. Results. Three distinct DNA species of different molecular sizes were generated from each engineered construct: the input construct; a unit-length chimeric PCV1Rep/PCV2Cap genome with a composite Ori but lacking the plasmid vector; and a molecule consisting of the remaining 0.75 copy PCV1Cap /PCV2Rep genome with a different composite Ori together with the bacterial plasmid. 4. Discussion/Conclusion. Replication of the input construct was presumably via the theta-replication mechanism utilizing the ColE1 Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria.