Functional characterization of the Rab11 and its response to SGIV infection in the orange-spotted grouper...

Rab11, a member of the Rab GTPase family, serves as a key regulator of vesicular trafficking and modulates viral infection in mammals, yet its functional role in aquatic viral infection remains largely uncharacterized. Singapore grouper iridovirus (SGIV), a member of the genus Ranavirus in the family Iridoviridae, is one of the most important viral pathogens afflicting grouper aquaculture. In this study, we investigated the influence of a novel Rab11 homolog (EcRab11) from the orange-spotted grouper (Epinephelus coioides) on SGIV infection and the role in host innate immune responses. EcRab11 shares high amino acid sequence identity with Rab11 orthologs spanning from mammals to teleost fishes, indicating evolutionary conservation of this GTPase. In healthy groupers, EcRab11 was predominantly expressed in major immune organs including the liver and spleen, and its transcript levels were significantly upregulated following immune challenges. Confocal imaging revealed that EcRab11 localizes to the cytoplasm as distinct punctate and vesicle-like structures. In contrast, overexpression of a constitutively active EcRab11 mutant (CA-EcRab11, with a GTP-locked mutation at the conserved Q70L site that renders the protein persistently activated and unable to hydrolyze GTP to GDP) induced the formation of enlarged vesicular structures, whereas overexpression of a dominant-negative EcRab11 mutant (DN-EcRab11, with a GDP-locked mutation at the conserved S25N site that abrogates GTP binding and locks the protein in an inactive state) resulted in a marked reduction in vesicle formation. Co-localization analyses further demonstrated that EcRab11 extensively colocalizes with the Golgi apparatus, early endosomes and late endosomes, and partially with the endoplasmic reticulum or lysosomes, but did not colocalize with mitochondria. Functional assays revealed that EcRab11 overexpression significantly suppressed SGIV infection, whereas overexpression of either CA-EcRab11 or DN-EcRab11 enhanced viral replication. Moreover, EcRab11 was found to positively regulate both autophagy and IFN immune responses in grouper cells. Taken together, our findings demonstrate that EcRab11 modulates SGIV infection by orchestrating host innate immune responses, thereby uncovering a novel antiviral mechanism of Rab11 against SGIV and providing new insights for the development of antiviral strategies for grouper aquaculture.