title: 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: Key Laboratory of Animal Virology, Ministry of Agriculture, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.
authors: Jia-Ling
authors: JL
authors: He
authors: Dan
authors: D
authors: Dai
authors: Niu
authors: N
authors: Zhou
authors: Ji-Yong
authors: JY
authors: Zhou
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D017925:Circovirus / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli; D005814:Genes, Viral; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D013552:Swine; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D014771:Virion / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication / Q000235:genetics
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D007074:Immunoglobulin G; D014764:Viral Proteins