Characterization of SNX5 in Orange-Spotted Grouper (Epinephelus coioides) during In Vitro Viral Infection

Abstract

SNX5 is a protein that is involved in endosomal sorting, signal transduction and endocytosis pathways. However, the roles of fish SNX5 were largely unknown. In this study, we identified an SNX5 homolog (EcSNX5) from an orange-spotted grouper (E. coioides) and investigated its role during viral infection. EcSNX5 encoded 412 amino acids with a PX domain and a BAR domain. In addition, it shared high identities with other known fish SNX5. Through quantitative real-time polymerase chain reaction (qRT-PCR), the high expression of EcSNX5 was observed in the head, kidney and heart. After stimulation with the red-spotted grouper nervous necrosis virus (RGNNV) in vitro, EcSNX5 expression was significantly induced. After RGNNV infection in vitro, EcSNX5 overexpression enhanced the expression of RGNNV genes, including coat protein (CP) and RNA-dependent RNA polymerase (RdRp). EcSNX5 knockdown downregulates expression of CP and RdRp. The TCID50 assay showed a higher viral titer when EcSNX5 is over expressed. Moreover, EcSNX5 overexpression could reduce the expression of interferon genes (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5 and TRIF) and inflammatory genes (IL6, IL8, IL-1β and TNF-α). EcSNX5 knockdown could promote the expression of interferon factors and inflammatory factors. Moreover, EcSNX5 overexpression suppresses the expression of autophagy genes (LC3-II, BECN1, ATG5 and ATG16L1) and upregulates the expression of apoptosis genes (Bax, BNIP3), but EcSNX5 knockdown had the opposite effect. According to the subcellular localization, EcSNX5 is localized in the cytoplasm and co-localizaed with RGNNV CP protein. The results showed EcSNX5 can influence viral infections by regulating the expression of interferon factors and inflammatory factors as well as adjusting virus-induced autophagy. These data will contribute to a better understanding of the immune response of fish during virus infection.