Nervous necrosis virus (NNV) can infect many species of fish and causes serious acute or persistent infection. However, its pathogenic mechanism is still far from clear. Specific cellular surface receptors are crucial determinants of the species tropism of a virus and its pathogenesis. Here, the heat shock protein 90ab1 of marine model fish species marine medaka (MmHSP90ab1) was identified as a novel receptor of red-spotted grouper NNV (RGNNV). MmHSP90ab1 interacted directly with RGNNV capsid protein (CP). Specifically, MmHSP90ab1 bound to the linker region (LR) of CP through its NM domain. Inhibition of MmHSP90ab1 by HSP90-specific inhibitors or MmHSP90ab1 siRNA caused significant inhibition of viral binding and entry, whereas its overexpression led to the opposite effect. The binding of RGNNV to cultured marine medaka hMMES1 cells was inhibited by blocking cell surface-localized MmHSP90ab1 with anti-HSP90β antibodies or pretreating virus with recombinant MmHSP90ab1 or MmHSP90ab1-NM protein, indicating MmHSP90ab1 was an attachment receptor for RGNNV. Furthermore, we found that MmHSP90ab1 formed a complex with CP and marine medaka heat shock cognate 70, a known NNV receptor. Exogenous expression of MmHSP90ab1 independently facilitated the internalization of RGNNV into RGNNV impenetrable cells (HEK293T), which was blocked by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Further study revealed that MmHSP90ab1 interacted with the marine medaka clathrin heavy chain. Collectively, these data suggest that MmHSP90ab1 is a functional part of the RGNNV receptor complex and involved in the internalization of RGNNV via the clathrin endocytosis pathway. Nervous necrosis virus (NNV), a Betanodavirus belonging to the Nodaviridae family, causes severe financial losses to aquaculture industry worldwide, nevertheless, its pathogenic mechanism remains unknown. Virus receptors are pivotal for viruses to cause infection and pathogenicity. However, knowledge regarding the cellular receptor for NNV is limited, and it has been unclear how the receptor participates in intracellular trafficking of NNV particles. In this study, we demonstrated that marine medaka heat shock protein 90ab1(MmHSP90ab1) was a functional receptor of RGNNV for the first time. Furthermore, MmHSP90ab1 expression could independently lead to RGNNV internalization through the clathrin endocytosis pathway. Our findings will contribute to developing novel strategies against NNV infection by targeting the MmHSP90ab1-clathrin endocytosis pathway.

Gametogenesis is a complicated biological process by which sperm and egg are produced for genetic transmission between generations. In many animals, the germline is segregated from the somatic lineage in early embryonic development through the specification of primordial germ cells (PGCs), the precursors of gametes for reproduction and fertility. In some species, such as fruit fly and zebrafish, PGCs are determined by the maternally provided germ plasm which contains various RNAs and proteins. Here, we identified a germ plasm/PGC-specific microRNA miR-202-5p for the first time in zebrafish. MiR-202-5p was specifically expressed in gonad. In female, it was expressed and accumulated in oocytes during oogenesis. Quantitative reverse transcription PCR and whole mount in situ hybridization results indicated that miR-202-5p exhibited a typical germ plasm /PGC-specific expression pattern throughout embryogenesis, which was consistent with that of the PGC marker vasa, indicating that miR-202-5p was a component of germ plasm and a potential PGC marker in zebrafish. Our present study might be served as a foundation for further investigating the regulative roles of miRNAs in germ plasm formation and PGC development in zebrafish and other teleost.

RIG-I-like receptors (RLRs) play important roles in response to virus infection by regulating host innate immune signaling pathways. Meanwhile, the RLR signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. Here, we found that zebrafish TRIM25 (zbTRIM25) functioned as a positive regulator of RLR signaling pathway during red spotted grouper nervous necrosis virus (RGNNV) infection. Post-RGNNV infection, zbTRIM25 expression was obviously inhibited and ectopic expression of zbTRIM25 led to enhanced expression of RLR signaling pathway-related genes. Overexpression and knockdown analysis revealed that zbTRIM25 promoted zebrafish RIG-I (zbRIG-I)-mediated IFN signaling and inhibited RGNNV replication. Mechanistically, zbTRIM25 bound to zbRIG-I; in particular, the SPRY domain of zbTRIM25 interacted with the tandem caspase activation and recruitment domains (2CARD) and repressor domain (RD) regions of zbRIG-I. zbTRIM25 promoted the K63 polyubiquitination of 2CARD and RD regions of zbRIG-I. Furthermore, zbTRIM25-mediated zbRIG-I activation of IFN production was enhanced by K63-linked ubiquitin, indicating that zbTRIM25-mediated zbRIG-I polyubiquitination was essential for RIG-I-triggered IFN induction. In conclusion, these findings reveal a novel mechanism that zbTRIM25 positively regulates the innate immune response by targeting and promoting the K63-linked polyubiquitination of zbRIG-I.

RIG-I–like receptor (RLR)–mediated antiviral signaling is critical to trigger the immune response to virus infection; however, the antiviral responses are also tightly regulated to avoid uncontrolled production of type I IFN by various mechanisms, including ubiquitination. In this study, an E3 ubiquitin ligase ring finger protein 114 (RNF114) from sea perch (Lateolabrax japonicus) (LjRNF114) was identified as a suppressor of RLR signaling pathways during red-spotted grouper nervous necrosis virus (RGNNV) infection. RGNNV infection promoted the expression of LjRNF114. Overexpression of LjRNF114 enhanced RGNNV replication, whereas knockdown of LjRNF114 led to opposite effects. Type I IFN production induced by RGNNV was suppressed by LjRNF114, which is dependent on its ubiquitin ligase activity. Moreover, LjRNF114 inhibited IFN promoter activation induced by key signaling molecules in RLR signaling pathways. We observed the interactions between LjRNF114 and both sea perch mitochondrial antiviral signaling protein (MAVS) and TNFR-associated factor 3 (TRAF3). Domain mapping experiments indicated that the RING and ubiquitin interacting motif domains of LjRNF114 were required for its interaction with TRAF3 and MAVS. We found that LjRNF114 targeted MAVS and TRAF3 for K27- and K48-linked ubiquitination and degradation, resulting in the inhibition of IFN production. Taken together, our study reveals, to our knowledge, a novel mechanism that LjRNF114 targets and promotes K27- and K48-linked ubiquitination of MAVS and TRAF3 to negatively regulate the RLR signaling pathways, promoting viral infection. Key Points LjRNF114 was a suppressor of RLR signaling pathways. LjRNF114 targeted and promoted K27- and K48-linked ubiquitination of MAVS and TRAF3.