Grouper Rab1 inhibits nodovirus infection by affecting virus entry and host immune response

Abstract

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens, respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo, implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies.