Analysis of putative ORF3 gene within porcine circovirus type 2.

Abstract

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic syndrome in pigs, whereas the ubiquitous related porcine circovirus type 1 (PCV1) is nonpathogenic. Corroborating an earlier observation in PCV2, Rep and Rep' proteins encoded by ORF1 are essential for the initiation of PCV2 replication. Cap protein encoded by ORF2 has a potential causative role in the initiation of PCV2 replication and contains a type-specific epitope. The putative ORF3 of PCV2 oriented in the opposite direction within ORF1 is unknown. In this study, ORF3-encoding protein of PCV2 was expressed in vitro as a fusion protein (GST-ORF3 protein), and monoclonal antibodies (MAbs) to the PCV2-ORF3-encoding protein were generated and biologically characterized. The mRNA transcript of ORF3 was characterized during a productive infection in PK-15 cells, and the PCV2 infectious DNA clone lacking ORF3 was constructed. GST-ORF3 protein, with an approximate molecular weight of 37.7 kDa, was obtained from the Escherichia coli transformed with the recombinant vector pGEX-4T-1-F3 after codon optimization of ORF3 DNA sequence. Four MAbs reacted strongly to the ORF3-encoding protein expressed in PK-15 cells in immunohistochemical staining. The mRNA transcript of ORF3 was confirmed in RT-PCR, Northern blot, and sequencing analyses. The progeny PCV2 virions were not revealed in the PK-15 cells transfected by the PCV2 infectious DNA clone without ORF3. These results demonstrate that the ORF3 of PCV2 can be transcribed and expressed and that ORF3-encoding protein plays a pivotal role in viral replication.

• Authors: Jia-Ling He, Dan Dai, Niu Zhou, Ji-Yong Zhou
• Journal: Hybridoma (2005) (Hybridoma (Larchmt)) Vol. 31 Issue 3 Pg. 180-7 (Jun 2012) ISSN: 1557-8348 [Electronic] United States
• PMID: 22741582 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal, Murine-Derived
• Antibodies, Neutralizing
• Antibodies, Viral
• Immunoglobulin G
• Viral Proteins

Topics

• Animals
• Antibodies, Monoclonal, Murine-Derived (biosynthesis, immunology)
• Antibodies, Neutralizing (biosynthesis, immunology)
• Antibodies, Viral (biosynthesis, immunology)
• Antibody Specificity
• Cell Line
• Circovirus (genetics, physiology)
• Escherichia coli
• Genes, Viral
• Immunoglobulin G (biosynthesis, immunology)
• Mice
• Mice, Inbred BALB C
• Mutagenesis, Site-Directed
• Swine
• Transcription, Genetic
• Viral Proteins (biosynthesis, genetics, immunology, physiology)
• Virion (genetics, physiology)
• Virus Replication (genetics)