Detection of rampant nucleotide reversion at the origin of DNA replication of porcine circovirus type 1

Mutational analysis was conducted to investigate the involvement of the “loop-sequence” (which is flanked by a pair of 11-nucleotide inverted repeats) at the origin of DNA replication of porcine circovirus type 1 with respect to viral protein synthesis, DNA self-replication and progeny virus production. The results demonstrated that an octanucleotide (A1G2T3A4T5T6A7C8) embedded in the loop is essential for viral DNA replication. Similar to previous work with porcine circovirus type 2, this octanucleotide can be further condensed to an essential core element represented by AxTAxTAC. After transfection, mutations introduced into the positions indicated by x (positions 2 and 5) were retained in the progeny viruses, while mutations engineered into the positions specified by the indicated nucleotides either did not yield any progeny virus (positions 6, 7, and 8) or they reverted back to wild-type nucleotide to generate infectious progeny viruses (positions 1, 3, and 4). In comparison to porcine circovirus type 2, porcine circovirus type 1 mutant genomes with perturbed octanucleotide sequences exhibited higher propensity to revert to wild-type under similar experimental conditions. The rate and frequency at which some of the nucleotide reversions occurred suggest that base complementarity may not be the governing factor for nucleotide incorporation at the porcine circovirus origin of DNA replication, and that the Rep-associated protein(s) may play a critical role in this process.