title: ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.
abstract: Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants.
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors: Zhangzhao
authors: Z
authors: Gao
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors: Qinfang
authors: Q
authors: Dong
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China. Electronic address: yonghoujiang@zstu.edu.cn.
authors: Yonghou
authors: Y
authors: Jiang
authors: The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian EH25 9RG, UK; Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, USA.
authors: Tanja
authors: T
authors: Opriessnig
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors: Jingxiu
authors: J
authors: Wang
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors: Yanping
authors: Y
authors: Quan
authors: College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors: Zongqi
authors: Z
authors: Yang
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D017925:Circovirus / Q000235:genetics* / Q000502:physiology*; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D013552:Swine; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D018389:Codon, Nonsense; D050505:Mutant Proteins; D012333:RNA, Messenger; D014764:Viral Proteins
keywords: Apoptosis; ORF4-protein deficient PCV2; Open reading frame 4; Porcine circovirus 2; Replication