Mutational analysis of the direct tandem repeat sequences at the origin of DNA replication of porcine circovirus type 1.

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.