Effects of transcription units and a specific hexanucleotide motif of porcine circovirus-like virus P1 on viral DNA synthesis, viral protein expression, and progeny virus production

Porcine circovirus-like virus P1 has a broad host range and is known to cause post-weaning multisystemic wasting syndrome in pigs, as well as infect and propagate in cattle, goats, dogs, cats, yaks, and rabbits. To investigate the functional importance of its eight open reading frames (ORFs) and the conserved hexanucleotide motif (GGATCC), we generated a panel of site-directed mutants based on the P1 infectious clone rpSK-2P1. These mutants were transfected into PK-15 cells and serially passaged four times. Viral replication capacity and genetic stability were assessed using polymerase chain reaction (PCR), quantitative PCR, and Sanger sequencing. All the mutant viruses retained the ability to replicate their genomes and transcribe viral RNA. However, sequencing of viral genomes recovered at the fourth passage revealed selective reversion at multiple mutated sites, indicating a strong replicative advantage conferred by wild-type sequences during sustained propagation. Notably, mutant Y1, which harbored a mutation in ORF1 that encoded the capsid protein (Cap), initially exhibited reduced replication but rapidly reverted upon passaging, restoring Cap expression and fitness. These findings demonstrate that although these ORFs and nucleotide sites do not abolish P1 replication, their native sequences play pivotal roles in modulating replication efficiency and maintaining genetic stability, thereby providing key insights into the molecular mechanisms governing P1 replication. Thus, P1 tolerates limited genetic change but is under intense pressure to maintain its wild-type sequence, offering critical insight into the replication strategy and evolutionary constraints of this compact, broad-host-range DNA virus.