Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2003
Publication Date: 5/1/2004
Citation: Cheung, A.K. 2004. Identification of the essential and non-essential transcription units for protein synthesis, DNA replication and infectious virus production of porcine circovirus type 1. Archives of Virology. 149(5):975-988. Interpretive Summary: Porcine circovirus type 2 (PCV2) is a newly emerged viral pathogen of swine. While clinical signs of disease and postmortem lesions induced by PCV2 are known, there is little information on the temporal pathogenesis and epidemiology of the virus. Because PCV2 was identified only recently, basic knowledge of the genetic and biochemical nature of the virus is limited. Standardized diagnostic tests have not developed and vaccines are not available. In previous work, we examined PCV2 RNA biosynthesis in tissue culture cells and identified several new PCV2 genetic elements that are different from the non-pathogenic PCV type 1 (PCV1), and we have determined the essential and non-essential transcription units required for PCV2 replication. In this work, we determined the essential and non-essential transcription units for PCV1 replication. Thus, this work provides a general frame work to elucidate the genetic basis for PCV1 and PCV2.
Technical Abstract: A plasmid-based transfection system capable of yielding infectious porcine circovirus type 1 (PCV1) was established and mutational analysis was conducted to investigate the involvement of each viral transcription unit in protein synthesis, DNA replication and progeny virus production. During PCV1 replication in PK15 cells, twelve viral-specific RNAs are synthesized. They include the capsid protein RNA (CR), eight Rep-associated RNAs (Rep, Rep', Rep3a, Rep3b, Rep3c-1, Rep3c-2, Rep3c-3 and Rep3c-4), and three NS-associated RNAs (NS462, NS642 and NS0). A stop codon introduced at the 5'-end of CR did not affect Rep-associated antigens or viral DNA synthesis. Altering the consensus dinucleotide at the splice junctions of the minor RNAs (Rep3a, Rep3b, Rep3c-1, Rep3c-2, Rep3c-3, Rep3c-4 and NS462) or introducing an early termination codon in Rep3c-4 and NS0 did not have any affect on viral protein synthesis, DNA replication or infectious virus production. However, mutations that resulted in a truncated Rep or Rep' or a modified P-loop Rep protein caused greater than 99% reduction of protein synthesis and complete shut down of viral DNA replication. Although NS642 could not be assayed in this study because it does not appear to code for any protein and silent mutation at the splice junction was not possible, the results here, together with those obtained from previous in vitro studies (11), demonstrated that only two proteins, Rep and Rep', are essential for PCV1 protein, DNA and infectious virus biosynthesis.