title: Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.
abstract: Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.
authors.forename: Chien-Wen
authors.initials: CW
authors.lastname: Chen
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Ming-Shan
authors.initials: MS
authors.lastname: Wu
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
authors.forename: Yi-Jen
authors.initials: YJ
authors.lastname: Huang
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Pei-Wen
authors.initials: PW
authors.lastname: Lin
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.
authors.forename: Chueh-Ju
authors.initials: CJ
authors.lastname: Shih
authors.affiliation: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Fu-Pang
authors.initials: FP
authors.lastname: Lin
authors.affiliation: Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan; Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
authors.forename: Chi-Yao
authors.initials: CY
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D053468:CARD Signaling Adaptor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D017994:Iridovirus / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010473:Perciformes / Q000821:virology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D053468:CARD Signaling Adaptor Proteins; D011500:Protein Synthesis Inhibitors; D014764:Viral Proteins; D003513:Cycloheximide; D053181:Caspase 8; D053453:Caspase 9