Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways

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.虹彩病毒科（Iridoviridae）蛙病毒属（Ranavirus）的石斑鱼虹彩病毒（Grouper iridovirus, GIV），其基因组携带有一段抗凋亡半胱天冬酶募集结构域（caspase recruitment domain, CARD）基因。GIV-CARD基因可编码一条由91个氨基酸组成的蛋白质，分子质量为10505道尔顿，与其他病毒CARD基因以及人类ICEBERG蛋白具有较高的序列相似性。本研究采用Northern印迹（Northern blot）技术证实，GIV-CARD的转录始于感染后4小时；进一步研究发现，其转录可被环己酰亚胺（cycloheximide）完全抑制，但不受阿非迪霉素（aphidicolin）影响，表明GIV-CARD属于早期基因。将GIV-CARD分别与增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）、FLAG标签融合的重组蛋白，可从细胞质转运至细胞核，但GIV-CARD序列中未发现典型的核定位信号（nuclear localization sequence）。在感染GIV的GK细胞中，通过RNA干扰（RNA interference）敲低GIV-CARD的表达，可抑制感染早期GIV-CARD及其他5种病毒基因的转录，并降低GIV的感染能力。免疫染色结果显示，表达GIV-CARD的细胞可有效抑制细胞凋亡。经紫外线照射或抗Fas抗体处理的HeLa细胞，可分别通过内源性和外源性凋亡通路发生凋亡；但在HeLa细胞中过表达重组GIV-CARD蛋白，可抑制线粒体通路及死亡受体通路介导的细胞凋亡。此外，本研究还发现，在抗Fas抗体诱导凋亡的HeLa细胞中，GIV-CARD的表达可显著降低半胱天冬酶-8（caspase-8）与半胱天冬酶-9（caspase-9）的活性。综上，上述结果证实GIV-CARD可同时通过内源性与外源性凋亡通路抑制细胞凋亡。