Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China; Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
Fish Shellfish Immunol. 2020 Sep;104:172-181. doi: 10.1016/j.fsi.2020.06.001. Epub 2020 Jun 10.
Interferon-induced transmembrane proteins (IFITMs) have been identified as important host restriction factors in mammals for the control of infection by multiple viruses. However, the antiviral functions of IFITMs against fish viruses remain largely uncertain. In this study, the IFITM3 homolog from orange spotted grouper (EcIFITM3) was cloned and its roles in grouper virus infection were investigated. The full-length cDNA of EcIFITM3 was 737 bp, which was composed of a 16 bp 5'-UTR, a 274 bp 3'-UTR, and a 447 bp ORF. EcIFITM3 encodes a 148-amino-acid polypeptide, which contains five domains, i.e., the N-terminal domain (aa 1-65), TM1 (aa 66-90), the cytoplasmic domain (aa 91-110), TM2 (aa 111-140), and the C-terminal domain (aa 141-148), and shares 78% and 47% identity with IFITM3 of gilthead seabream (Sparus aurata) and human (Homo sapiens), respectively. EcIFITM3 mRNA was detected in 12 tissues of healthy groupers, with the highest expression levels in the head kidney. Additionally, the in vitro mRNA levels of EcIFITM3 were significantly upregulated by infection with Singapore grouper iridovirus (SGIV) or red spotted grouper nervous necrosis virus (RGNNV), or treatment with polyinosinic-polycytidylic acid (poly I:C) or lipopolysaccharide (LPS). Subcellular localization analysis showed that EcIFITM3 was mainly distributed in the cell membrane of grouper cells. In vitro, the ectopic expression of EcIFITM3 inhibited SGIV and RGNNV infection, as demonstrated by the reduced severity of the cytopathic effect, decreased virus production, and low levels of viral mRNA and proteins. Consistently, knockdown of EcIFITM3 by small interfering RNAs (siRNAs) enhanced SGIV and RGNNV replication. EcIFITM3 overexpression and knockdown experiments both suggested that EcIFITM3 inhibits the infection of SGIV and RGNNV by restricting virus entry.
干扰素诱导跨膜蛋白（IFITMs）已被鉴定为哺乳动物中控制多种病毒感染的重要宿主限制因子。然而，IFITMs 对鱼类病毒的抗病毒功能在很大程度上仍不确定。在这项研究中，我们克隆了橙色斑点石斑鱼（EcIFITM3）的 IFITM3 同源物，并研究了其在石斑鱼病毒感染中的作用。EcIFITM3 的全长 cDNA 为 737bp，由 16bp 的 5'-UTR、274bp 的 3'-UTR 和 447bp 的 ORF 组成。EcIFITM3 编码一个由 148 个氨基酸组成的多肽，含有 5 个结构域，即 N 端结构域（aa1-65）、TM1（aa66-90）、细胞质结构域（aa91-110）、TM2（aa111-140）和 C 端结构域（aa141-148），与金头鲷（Sparus aurata）和人类（Homo sapiens）的 IFITM3 分别具有 78%和 47%的同源性。EcIFITM3 mRNA 在健康石斑鱼的 12 种组织中均有检测到，在头肾中的表达水平最高。此外，EcIFITM3 的体外 mRNA 水平在感染新加坡石斑鱼虹彩病毒（SGIV）或红鳍东方鲀神经坏死病毒（RGNNV），或用多聚肌苷酸-多聚胞苷酸（poly I:C）或脂多糖（LPS）处理后显著上调。亚细胞定位分析表明，EcIFITM3 主要分布在石斑鱼细胞的细胞膜上。在体外，EcIFITM3 的异位表达抑制了 SGIV 和 RGNNV 的感染，表现为细胞病变效应的严重程度降低、病毒产量减少以及病毒 mRNA 和蛋白水平降低。一致地，通过小干扰 RNA（siRNAs）敲低 EcIFITM3 增强了 SGIV 和 RGNNV 的复制。EcIFITM3 的过表达和敲低实验均表明，EcIFITM3 通过限制病毒进入来抑制 SGIV 和 RGNNV 的感染。
