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United States Department of Agriculture

Agricultural Research Service

Title: MUTATIONAL ANALYSIS OF THE DIRECT TANDEM REPEAT SEQUENCES AT THE ORIGIN OF DNA REPLICATION OF PORCINE CIRCOVIRUS TYPE 1

Author
item Cheung, Andrew

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 26, 2005
Publication Date: September 1, 2005
Citation: Cheung, A.K. 2005. Mutational analysis of the direct tandem repeat sequences at the origin of DNA replication of porcine circovirus type 1. Virology. 339:192-199.

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 based on substitution mutagenesis. In this work, we examined the mechanisms involved in PCV2 DNA replication and identified a 6-nucleotide tandem sequence that is essential for viral protein, DNA and progeny virus biosynthesis. Thus, this work provides insight into the life-cycle of PCV and a general frame work to generate attenuated viruses. The information obtained advances our understanding of circovirus biology and aids the research of scientists in industry, universities and government agencies.

Technical Abstract: Mutational analysis was conducted to investigate the role of the nucleotide sequences flanking the stem-loop palindromic structure at the origin of DNA replication of porcine circovirus type 1 (PCV1) with respect to self-DNA replication and progeny virus generation. The results demonstrated that the A-rich sequence to the left of the palindrome is non-essential for virus replication. Although a set of four hexanucleotide (H) sequences to the right of the palindrome (organized in two tandem repeats: the proximal H1/H2 and the distal H3/H4) are binding-sites for the viral Rep-associated proteins in vitro, only a proximal tandem (H/H or h-like/H) is essential for PCV1 DNA replication. In the presence of H1/H2, mutations engineered into H3/H4 were preserved in the progeny viruses. Mutations engineered into H1/H2 were invariably deleted so that the downstream H3/H4 was placed next to the palindrome. Viral genome with mutations engineered into both H1/H2 and H3/H4 underwent extensive nucleotide reorganization to yield progeny viruses containing either H3/H4, h-like/H4, or h-like/H3/H4 sequences.

Last Modified: 10/31/2014
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