Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2006
Publication Date: 9/1/2006
Citation: Cheung, A.K. 2006. Rolling-circle replication of an animal circovirus genome in a Theta-replicating bacterial plasmid in Escherichia coli. Journal of Virology. 80(17):8686-8694. Interpretive Summary: Porcine circovirus type 2 (PCV2) is a newly emerged viral pathogen of swine. While clinical signs of disease and postmortem lesions induced by PCV2 are known, there is little information on the temporal pathogenesis and epidemiology of the virus. Standardized diagnostic tests have not developed and vaccines are not available. In previous work, we examined the genetic elements synthesized by PCV type 1 (PCV1) and PCV2 in tissue culture cells. We have identified several new PCV2 genetic elements that are different from the non-pathogenic PCV1 and have determined the essential and non-essential genetic elements required for PCV1 and PCV2 replication. We also proposed a novel rolling circle "melting-pot" model to account for its replication. In this work, we discovered that PCV DNA replication can occur in E. coli. Thus, this work provides insight into the origin of PCV and a general frame work to generate recombinant viruses more efficiently. The information obtained advances our understanding of circovirus biology and aids the research of scientists in industry, universities and government agencies.
Technical Abstract: A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem [O.8 copy of PCV type 1 (PCV1), 0.95 copy of PCV type 2 (PCV2)] with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct; a unit-length chimeric PCV1Rep/PCV2Cap genome with a composite Ori but lacking 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. 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. The fact that the Rep’ protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestion that circular single-stranded DNA animal circoviruses, plant geminiviruses and nanoviruses may have evolved from prokaryotic episomal replicons.