College of Marine Sciences, South China Agricultural University, Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
College of Marine Sciences, South China Agricultural University, Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China; Southern Marine Science and Engineering Guangdong Laboratory, Zhuhai, 528478, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266000, China.
Fish Shellfish Immunol. 2022 Nov;130:43-52. doi: 10.1016/j.fsi.2022.09.003. Epub 2022 Sep 6.
Cystatin F (CyF), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyF and its latent molecular mechanism during virus infection in fish remain vacant. In our research, we cloned the open reading frame (ORF) of CyF homology from orange-spotted grouper (Ec-CyF) consisting of 342 nucleotides and encoding a 114-amino acid protein. Ec-CyF included two cystatins family sequences containing one KXVXG sequence without the signal peptide, and a hairpin ring containing proline and tryptophan (PW). Tissue distribution analysis indicated that Ec-CyF was highly expressed in spleen and head kidney. Besides, further analysis showed that the expression of Ec-CyF increased during SGIV infection in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyF was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyF demoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was impeded, as well as the restraint of caspase 3/7 and caspase 8. In addition, Ec-CyF overexpression up-regulated the expression of IFN related molecules including ISG15, IFN, IFP35, IRF3, IRF7, MYD88 and down-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyF-overexpressing increased the activity of IFN3 and ISRE promoter, but impeded NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyF was involved in innate immunity response and played a key role in DNA virus infection.
半胱氨酸蛋白酶抑制剂 F（CyF）是一种免疫防御和癌症治疗领域广泛研究的物质。然而，在鱼类病毒感染过程中，CyF 的功能及其潜在的分子机制仍不清楚。在我们的研究中，我们从鞍带石斑鱼（Ec-CyF）中克隆了 CyF 同源物的开放阅读框（ORF），其由 342 个核苷酸组成，编码一个包含 114 个氨基酸的蛋白质。Ec-CyF 包含两个半胱氨酸蛋白酶抑制剂家族序列，其中一个序列含有不含信号肽的 KXVXG 序列，另一个序列含有脯氨酸和色氨酸（PW）组成的发夹环。组织分布分析表明，Ec-CyF 在脾脏和头肾中高度表达。此外，进一步的分析表明，Ec-CyF 在石斑鱼脾脏（GS）细胞感染 SGIV 时表达增加。亚细胞定位分析表明，Ec-CyF 在 GS 细胞中主要分布在细胞质中。过表达 Ec-CyF 降低了病毒基因 MCP、VP19 和 LITAF 的 mRNA 水平。同时，脂肪头鲦（FHM）细胞中的 SGIV 诱导凋亡受到抑制，半胱天冬酶 3/7 和半胱天冬酶 8 的活性也受到抑制。此外，Ec-CyF 过表达上调了 IFN 相关分子的表达，包括 ISG15、IFN、IFP35、IRF3、IRF7、MYD88，并下调了促炎因子如 IL-1β、IL-8 和 TNF-α的表达。同时，Ec-CyF 过表达增加了 IFN3 和 ISRE 启动子的活性，但通过荧光素酶报告基因检测抑制了 NF-κB 启动子的活性。综上所述，我们的研究结果表明，Ec-CyF 参与了先天免疫反应，并在 DNA 病毒感染中发挥了关键作用。
