Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2005
Publication Date: 3/1/2006
Citation: Cheung, A.K. 2005. Regeneration of the replication-associated proteins tandem direct repeat recognition nucleotide sequence at the origin of DNA replication of porcine circovirus type 1. Virology. 346(1):32-39. Interpretive Summary: Porcine circovirus type 2 (PCV2) is a newly emerged viral pathogen of swine that is associated with a wasting syndrome in weaned pigs. While clinical signs of disease and postmortem lesions associated with PCV2 are known, there is little information on the temporal pathogenesis and epidemiology of the virus. Standardized diagnostic tests have not been 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 “melting-pot” model to account for its replication. In this work, we examined the mechanisms involved in PCV1 DNA replication and identified a hexanucleotide direct repeat signature DNA 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 that might prove useful as vaccines for PCV or vaccine vectors for other viral diseases.
Technical Abstract: Four copies of a hexanucleotide (H) sequence are located to the right of the palindrome at the origin of DNA replication of the porcine circovirus type 1 (PCV1) genome. These sequences are organized in two direct tandems, the proximal H1/H2 and the distal H3/H4 repeats, and have been shown to be binding-sites for the essential Rep and Rep’ proteins. Previous work demonstrated that infectious PCV1 virion can accommodate a variable number of H sequences at the origin of DNA replication. In this work, mutational analysis was conducted to elucidate the critical core element within the hexanucleotide with respect to self-DNA replication and progeny virus synthesis. It was found that while a single H sequence abutting the palindrome is sufficient for PCV1 viability, a tandem repeat arrangement is the more stable and thus preferred configuration. Within the H sequence, selected nucleotides at specific positions are critical for Rep-associated proteins recognition and for viral DNA replication.