Wanwan Zhang

The CD40–CD154 receptor-ligand axis is a core costimulatory regulator of antiviral immunity in mammals, but its functional role in teleosts remains largely unknown. Here, we identified the CD40 and CD154 homologs (MsCD40 and MsCD154) from largemouth bass (Micropterus salmoides), a globally farmed perciform teleost. Bioinformatic analysis confirmed that MsCD40 and MsCD154 harbor the conserved domain architectures of tumor necrosis factor receptor superfamily and TNF superfamily, respectively, with a teleost-specific phylogenetic clustering pattern. Both genes were ubiquitously expressed in immune-relevant tissues, and their transcription was dynamically regulated in response to viral hemorrhagic septicemia virus (VHSV) and largemouth bass virus (LMBV) challenge in vivo. Co-immunoprecipitation and immunofluorescence co-localization assays verified that MsCD40 and MsCD154 physically interact at the plasma membrane, forming a functional receptor-ligand complex. Functional assays showed that overexpression of either MsCD40 or MsCD154 significantly suppressed VHSV and LMBV infection in vitro. Furthermore, MsCD40 and MsCD154 overexpression dose-dependently activated nuclear factor-κB (NF-κB) reporter activity, and markedly upregulated the transcription of NF-κB downstream effector genes, including IL-8, NLRP3 and P105, under both VHSV and LMBV infection. Collectively, our findings demonstrate that the teleost CD40–CD154 costimulatory axis restricts both RNA and DNA viral infection in largemouth bass through NF-κB-mediated immune activation, which provides promising molecular targets for the development of broad-spectrum antiviral strategies in largemouth bass aquaculture.

Rhabdoviruses exhibit a broad host range, yet the cellular receptors underlying their cross-species tropism remain poorly defined. Here, we identified tetraspanin CD9 as a conserved functional entry receptor for diverse rhabdoviruses across genera, including viral hemorrhagic septicemia virus (VHSV) ( Novirhabdovirus ), Siniperca chuatsi rhabdovirus (SCRV) ( Siniperhavirus ), and vesicular stomatitis virus (VSV) ( Vesiculovirus ). We demonstrated that the domain IV of VHSV glycoprotein G directly interacted with the large extracellular loop domain of Lateolabrax japonicus CD9 (LjCD9). CD9 knockout, CD9 protein, or CD9 antibody significantly reduced VHSV infection in vitro and CD9 knockout zebrafish, while HEK293T cells, which are nonsusceptible but permissive to VHSV, become susceptible when expressing LjCD9, suggesting that LjCD9 is an entry receptor for VHSV. We further confirmed that LjCD9 functions as a functional receptor of SCRV. Importantly, the human CD9 orthologue can serve as a receptor of VSV. Our findings also revealed that LjCD9 mediated VHSV entry via clathrin- and caveolae-mediated endocytosis. Notably, nitazoxanide (NTZ) was identified as a broad-spectrum inhibitor of VHSV, SCRV, and VSV likely by interfering with the G protein–CD9 interaction. This study establishes CD9 as a cross-species receptor for rhabdoviruses and highlights NTZ as a promising broad-spectrum antiviral agent.

Nervous necrosis virus (NNV), a highly pathogenic RNA virus, is a major pathogen in the global aquaculture industry. To efficiently infect fish, NNV must evade or subvert the host IFN for their replication; however, the precise mechanisms remain to be elucidated. In this study, we reported that capsid protein (CP) of red-spotted grouper NNV (RGNNV) suppressed the IFN antiviral response to promote RGNNV replication in Lateolabrax japonicus brain cells, which depended on the ARM, S, and P domains of CP. CP showed an indirect or direct association with the key components of retinoic acid–inducible gene-I–like receptors signaling, L. japonicus TNFR-associated factor 3 (LjTRAF3) and IFN regulatory factor (LjIRF3), respectively, and degraded LjTRAF3 and LjIRF3 through the ubiquitin-proteasome pathway in HEK293T cells. Furthermore, we found that CP potentiated LjTRAF3 K48 ubiquitination degradation in a L. japonicus ring finger protein 114–dependent manner. LjIRF3 interacted with CP through the S domain of CP and the transcriptional activation domain or regulatory domain of LjIRF3. CP promoted LjIRF3 K48 ubiquitination degradation, leading to the reduced phosphorylation level and nuclear translocation of LjIRF3. Taken together, we demonstrated that CP inhibited type I IFN response by a dual strategy to potentiate the ubiquitination degradation of LjTRAF3 and LjIRF3. This study reveals a novel mechanism of RGNNV evading host immune response via its CP protein that will provide insights into the complex pathogenesis of NNV. Key Points CP potentiates RGNNV proliferation by suppressing the RLR-IFN signaling pathway. CP promotes LjRNF114-mediated K48 ubiquitination and degradation of LjTRAF3. CP promotes the K48-linked ubiquitination and degradation of LjIRF3.

Upon virus invasion of the host, APCs process Ags to short peptides for presentation by MHC class II (MHC-II). The recognition of virus-derived peptides in the context of MHC-II by CD4⁺ T cells initiates the adaptive immune response for virus clearance. As a survival instinct, viruses have evolved mechanisms to evade Ag processing and presentation. In this study, we discovered that IFN-γ induced endogenous MHC-II expression by a sea perch brain cell line through the STAT1/IFN regulatory factor 1 (IRF1)/CIITA signaling pathway. Furthermore, viral hemorrhagic septicemia virus infection significantly inhibited the IFN-γ–induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-β genes. By contrast, although STAT1 transcript was upregulated, paradoxically, the STAT1 protein level was attenuated. Moreover, overexpression analysis revealed that viral hemorrhagic septicemia virus N protein blocked the IFN-γ–induced expression of IRF1, CIITA, MHC-II-α, and MHC-II-β genes, but not the STAT1 gene. We also found out that N protein interacted with STAT1 and enhanced the overall ubiquitination level of proteins, including STAT1 in Lateolabrax japonicus brain cells. Enhanced ubiquitination of STAT1 through K48-linked ubiquitination led to its degradation through the ubiquitin–proteasome pathway, thereby inhibiting the biological function of STAT1. Our study suggests that aquatic viruses target Ag presentation in lower vertebrates for immune evasion as do mammalian viruses. Key Points VHSV infection affects MHC-II expression induced by IFN-γ in sea perch. N protein of VHSV degrades STAT1 through K48-linked ubiquitination.

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.