Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural University, Guangzhou, China.
College of Fisheries, Henan Normal University, Xinxiang, China.
Front Immunol. 2020 Sep 2;11:2133. doi: 10.3389/fimmu.2020.02133. eCollection 2020.
The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, , and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to , and , respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions.
小 GTPase Rab5 是囊泡运输的主要调节因子之一，参与内吞作用的早期阶段，如内吞作用和内体成熟。三种 Rab5 同工型（a、b 和 c）具有高度的序列同一性，并表现出复杂的功能。然而，Rab5c 在病毒感染和细胞免疫反应中的作用仍知之甚少。在这项研究中，基于已建立的病毒-细胞感染模型，我们研究了 Rab5c 在病毒感染和宿主免疫反应中的作用。从橙色斑点石斑鱼中克隆了 Rab5c，并将其命名为 EcRab5c。EcRab5c 编码一个 220 个氨基酸的多肽，与 和 分别具有 99%和 91%的同一性。共聚焦成像显示 EcRab5c 在细胞质中定位于点状结构。然而，组成型激活（CA）EcRab5c 突变体导致囊泡增大，而显性负（DN）EcRab5c 突变体减少囊泡结构。SGIV 感染后 EcRab5c 的表达水平显著增加。EcRab5c 敲低或 CA/DN EcRab5c 过表达显著抑制 SGIV 感染。通过单颗粒成像分析，我们进一步观察到 EcRab5c 破坏在 SGIV 感染的早期阶段会损害关键事件，包括在单个病毒水平上的病毒结合、进入和从早期到晚期内体的运输。此外，这是首次研究 EcRab5c 在自噬中的作用。同样，EcRab5c 正向调节干扰素相关因子和促炎细胞因子。总之，这些数据表明 EcRab5c 在鱼类虹彩病毒感染和宿主免疫中发挥双重功能，进一步加深了我们对病毒和宿主免疫相互作用的理解。
