Fas and Fas ligand (FasL) pathway plays important roles in virus defense and cell apoptosis. In our previous work, nervous necrosis virus (NNV) was discovered in Pacific cod (Gadus macrocephalus), and the Fas ligand (PcFasL) was up-regulated when NNV outbreak, however, signal transmission of Fas/FasL in fish are still unclear. In the present study, Pacific cod Fas (PcFas), PcFasL and Fas-associating protein with a novel death domain (PcFADD) were characterized. The predicted protein of PcFas, PcFasL and PcFADD includes 333 aa, 90 aa and 93 aa, separately. 3-D models of PcFas, PcFasL and PcFADD were well constructed based on reported templates, respectively, even though the sequence homology with other fish is very low. The transcript levels of PcFas increased gradually from 15 day-post hatching (dph) to 75dph. PcFas was significantly up-regulated when cod larvae had NNV symptoms at 24dph, 37dph, 46dph, 69dph, and 77dph. Subcellular localization revealed that PcFasL was located in the cytoplasm, while PcFas was mainly located in the cell membrane. Exogenous expressed PcFasL of 900 μg/mL could kill the Epithelioma papulosum cyprinid (EPC) cells by MTT test, but low concentration has no effect on the cells. qPCR analysis showed that overexpression of PcFas could significantly up-regulate the expression of genes related to Fas/FasL signaling pathway, including bcl-2, bax, and RIP3, while overexpression of PcFasL significantly up-regulate the expression of caspase-3, caspase-9, and MLKL. Overexpression of PcFas or PcFasL could induce EPC apoptosis significantly by flow cytometry, which was consistent with the results of caspase-3 mRNA level increasing. The results indicated that NNV could induce apoptosis through Fas/FasL signal pathway.

Fas和Fas配体（FasL）通路在病毒防御和细胞凋亡中发挥重要作用。在我们之前的工作中，在太平洋鳕鱼（ Gadus macrocephalus）中发现了神经坏死病毒（NNV）。), 并且在 NNV 爆发时 Fas 配体 (PcFasL) 被上调, 然而, Fas/FasL 在鱼类中的信号传递仍不清楚。在本研究中，对太平洋鳕鱼 Fas (PcFas)、PcFasL 和具有新型死亡结构域的 Fas 相关蛋白 (PcFADD) 进行了表征。PcFas、PcFasL和PcFADD的预测蛋白分别包括333 aa、90 aa和93 aa。PcFas、PcFasL 和 PcFADD 的 3-D 模型分别基于报告的模板很好地构建，即使与其他鱼类的序列同源性非常低。PcFas 的转录水平从孵化后 15 天 (dph) 逐渐增加到 75dph。当鳕鱼幼虫在 24dph、37dph、46dph、69dph 和 77dph 出现 NNV 症状时，PcFas 显着上调。亚细胞定位显示 PcFasL 位于细胞质中，而PcFas主要位于细胞膜上。外源表达的 900 μg/mL 的 PcFasL 可以杀死MTT法检测上皮瘤（EPC）细胞，但低浓度对细胞无影响。qPCR分析表明，PcFas过表达可显着上调Fas/FasL信号通路相关基因的表达，包括bcl-2、bax和RIP3，而PcFasL过表达可显着上调caspase-3、caspase的表达。 -9 和 MLKL。流式细胞仪检测过表达PcFas或PcFasL可显着诱导EPC凋亡，这与caspase-3 mRNA水平升高的结果一致。结果表明NNV可通过Fas/FasL信号通路诱导细胞凋亡。