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United States Department of Agriculture

Agricultural Research Service

Title: A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication

Author
item Cheung, Andrew

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 17, 2007
Publication Date: June 20, 2007
Citation: Cheung, A.K. 2007. A stem-loop structure, sequence non-specific, at the origin of DNA replication of porcine circovirus is essential for termination but not for initiation of rolling-circle DNA replication. Virology. 363(1):229-235.

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. Recently, we discovered that PCV DNA replication can occur in E. coli. In this work, we examined the involvement of the "stem-loop" structure (at the origin of DNA replication) during the initiation and termination of replication process. 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 stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. In this work, a head-to-tail tandem construct of porcine circovirus capable of generating unit-length genomic DNA in Escherichia coli was employed to examine the role of the stem-loop structure with respect to the RCR initiation and termination process. The advantage of using a head-to-tail tandem construct is that the initiation and termination sites for generation of the unit-length viral genomes are physically separated, which allows independent examination of the initiation/termination processes. Nucleotide substitution mutational analysis showed that a pair of inverted repeats capable of forming a stem-loop structure was essential for termination, but not for initiation. The results also demonstrated that it is the stem-loop configuration, not nucleotide sequence specificity, which is critical for termination of RCR DNA replication.

Last Modified: 4/16/2014
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