Submitted to: International Congress of Virology
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2005
Publication Date: July 23, 2005
Citation: Cheung, A.K. 2005. Mutational analysis of the tandem direct repeats at the Ori of porcine circovirus [abstract]. International Congress of Virology. p. 79-80. Technical Abstract: Background The large intergenic region of PCV contains the Ori with multiple elements crucial for viral DNA replication. There is the 3'-portion of the Rep gene promoter, the octanucleotide motif that embeds the presumed cleavage-site for initiation of plus-strand DNA replication, the palindromic sequence that form the stem of the 4-stranded rolling circle melting-pot DNA replication model, the signal for termination of DNA replication, and the direct H repeats which are binding-sites for the essential Rep and Rep' proteins in vitro. In this work, we examined the importance of the immediate sequences flanking the palindromic stem-loop structure of the melting-pot: the A-rich sequence (on the left) and the hexanucleotide repeats, H1/H2 and H3/H4 (on the right) with respect to viral protein synthesis, self-DNA replication and progeny virus production. Methods A PCV1 genomic clone (J1), capable of producing infectious PCV1 upon transfection into PK15 cells after excision and re-circularization of the viral DNA was employed to construct the mutant genomes used in this study. At 48 h posttransfection, one set of transfected cultures was assayed for viral protein synthesis by immunochemical staining. At 7 days posttransfection, a second set of transfected cultures was assayed for infectious virus. At designated cell passage, viral DNAs were isolated for nucleotide sequence determination. Results (I) Mutagenesis of the A-rich sequence to the left of the stem-loop structure. Rep-positive cells (100% J1) were observed and progeny viruses retained the engineered mutations. (II) Mutagenesis of the H sequences to the right of the stem-loop structure. (i). Disruption of the distal H3/H4 tandem. Rep-positive cells were reduced and progeny viruses retained the input mutations. (ii). Disruption of the proximal H1/H2 tandem. Rep-positive cells were significantly reduced and progeny viruses have deleted the H1/H2 mutant sequence. (iii). Disruption of both the proximal H1/H2 and the distal H3/H4 tandem repeats. Rep-positive cells were significantly reduced. Progeny viruses were recovered occasionally and they exhibited modified tandem H motif sequences (h-like/H4). Conclusions 1. The A-rich sequence is non-essential for PCV replication. 2. The number of H sequences present in the infectious virus is flexible.