Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. ABSTRACT Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host’s immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish

ABSTRACT Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus , infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP’s inhibitory action was more pronounced than ProA’s protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV. IMPORTANCE As a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions. 

Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion

Methylation at the N6 position of adenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, tightly associating with regulation of viral life circles and immune responses. Here, a methyltransferase-like 3 homolog gene from sea perch (Lateolabrax japonicus), designated LjMETTL3, was cloned and characterized, and its negative role in fish virus pathogenesis was uncovered. The cDNA of LjMETTL3 encoded a 601-amino acid protein with a MT-A70 domain, which shared the closest genetic relationship with Echeneis naucrates METTL3. Spatial expression analysis revealed that LjMETTL3 was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. LjMETTL3 expression was significantly upregulated at 12 and 24 h post RGNNV and VHSV infection in vitro. In addition, ectopic expression of LjMETTL3 inhibited RGNNV and VHSV infection in LJB cells at 12 and 24 h post infection, whereas knockdown of LjMETTL3 led to opposite effects. Furthermore, we found that LjMETTL3 may participate in boosting the type I interferon responses by interacting with TANK-binding kinase. Taken together, these results disclosed the antiviral role of fish METTL3 against RGNNV and VHSV and provided evidence for understanding the potential mechanisms of fish METTL3 in antiviral innate immunity. 

Methyltransferase-like 3 suppresses red spotted grouper nervous necrosis virus and viral hemorrhagic septicemia virus infection by enhancing type I interferon responses in sea perch.

ABSTRACT Nervous necrosis virus (NNV), a highly contagious fish virus, has caused huge economic losses to the global aquaculture industry. A previous study showed that protein A (ProA) encoded by orange-spotted grouper NNV triggers type I interferon (IFN) production in fish cells, but the activation and modulation of correlative signal pathways remain unclear. Here, we proved that ProA induces fish cell-specific IFN promoter activation in a dose-dependent manner. In channel catfish ovary (CCO, an NNV-permissive cell), ProA evoked expression and secretion of functional IFN with anti-DNA virus activity, suggesting a good model for IFN signaling research. A contrastive study of signaling in CCO and fathead minnow (FHM, an NNV-nonpermissive cell) using RNAi knockdown or dominant negative mutant overexpression verified that ProA-mediated IFN activation went through retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway (RIG-I/MDA5-MAVS-TRAF3-TBK1-IRF3) while NOD1-RIPK2-NFκB and TLR3-TRIF branches were unnecessary. As RNA receptors upregulated by ProA in FHM, RIG-I and MDA5 promoted ProA-mediated IFN activation to form a positive feedback loop, while LGP2, NOD1, and PKR inhibited this activation as negative modulators. In ProA-expressed, NNV-infected CCO, the transcription of RIG-I, MDA5, MAVS, TBK1, IRF3, and IFN was downregulated, but the expression of LGP2, TRAF3, and NOD1 remained unchanged, suggesting unknown IFN suppression mechanism by NNV infection. IFN inhibition by overexpressing mutated RIG-I greatly enhanced NNV replication in FHM, implying that RIG-I might be the main target for both ProA-mediated activation and NNV infection-induced inhibition. This study provides overviews and foundations for understanding the interaction between betanodavirus-encoded protein and fish innate immune signaling. IMPORTANCE As a major pathogen, nervous necrosis virus (NNV) infects more than 120 fish species worldwide and is virulent to larvae and juvenile fish, hampering the development of the fish fry industry. Understanding virus-host interaction and underlying mechanisms is an important but largely unknown issue in fish virus studies. Here, using channel catfish ovary and fathead minnow cells as models for the study of innate immunity signaling, we found that NNV-encoded ProA activated interferon signaling via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway which was still suppressed by the infection of wild-type NNV. This finding has important implications for the comprehension of NNV protein function and the immune response from different cells. First, RIG-I is the key node for anti-NNV innate immunity. Second, the response intensity of RLR signaling determines the degree of NNV proliferation. This study expands our knowledge regarding the overview of signal pathways affected by NNV-encoded protein and also highlights potential directions for the control of aquatic viruses. As a major pathogen, nervous necrosis virus (NNV) infects more than 120 fish species worldwide and is virulent to larvae and juvenile fish, hampering the development of the fish fry industry. Understanding virus-host interaction and underlying mechanisms is an important but largely unknown issue in fish virus studies. Here, using channel catfish ovary and fathead minnow cells as models for the study of innate immunity signaling, we found that NNV-encoded ProA activated interferon signaling via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway which was still suppressed by the infection of wild-type NNV. This finding has important implications for the comprehension of NNV protein function and the immune response from different cells. First, RIG-I is the key node for anti-NNV innate immunity. Second, the response intensity of RLR signaling determines the degree of NNV proliferation. This study expands our knowledge regarding the overview of signal pathways affected by NNV-encoded protein and also highlights potential directions for the control of aquatic viruses.

Orange-spotted grouper nervous necrosis virus-encoded protein A induces interferon expression via RIG-I/MDA5-MAVS-TBK1-IRF3 signaling in fish cells

The battle between host and viral is ubiquitous across all ecosystems. Despite this, research is scarce on the antiviral characteristics of fish, particularly in those that primarily rely on innate immune responses. This study, comprehensively explored the genetic and antiviral features of ISG15 in spotted seabass, focusing on its response to largemouth bass ulcerative syndrome virus (LBUSV). Through whole-genome BLAST and PCR cloning, two ISG15 homologs, namely LmISG15a and LmISG15b, were identified in spotted seabass, both encoding highly conserved proteins. However, a distinctive contrast emerged in their expression patterns, with LmISG15a exhibiting high expression in immune organs while LmISG15b remained largely silent across various organs. Regulatory elements analysis indicated an asymmetric evolution of the two ISG15s, with the minimal expression of LmISG15b may attribute to the loss of a necessary ISRE and an additional instability "ATTTA" motif. Association analysis demonstrated a significant correlation between LmISG15a expression and LBUSV infection. Subsequent antiviral activity detection revealed that LmISG15a interacted with LBUSV, inhibiting its replication by activating ISGylation and downstream pro-inflammatory mediators. In summary, this study unveils a distinct evolutionary strategy of fish antiviral gene ISG15 and delineates its kinetic characteristics in response to LBUSV infection. 

Asymmetric evolution of ISG15 homologs and the immune adaptation to LBUSV infection in spotted seabass (Lateolabrax maculatus).

Nectin4 antagonises type I interferon production by targeting TRAF3 for autophagic degradation and disrupting TRAF3-TBK1 complex formation.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control on host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that fish pathogen nervous necrosis virus (NNV) recruited an E3 ubiquitin ligase ring finger protein 34 (RNF34) to inhibit RLRs-mediated interferons (IFN) response via ubiquitinating TBK1 and IRF3. Ectopic expression of RNF34 greatly enhances NNV replication and prevents IFN production, while deficiency of RNF34 led to the opposite effect. Furthermore, RNF34 targets TBK1 and IRF3 via its RING domain. Of note, the interactions between RNF34 and TBK1 or IRF3 were conserved in different fish species. Mechanically, RNF34 promote K27-linked ubiquitination and degradation of TBK1 and IRF3, which in turn diminishing TBK1-induced translocation of IRF3 from cytoplasm to nucleus. Ultimately, NNV capsid protein (CP) was found directly bind with RNF34 and this interaction was conserved in different fishes, and CP induced TBK1 and IRF3 degradation and IFN suppression was depended on RNF34. Our finding demonstrated a novel mechanism by which NNV CP evaded host innate immunity via RNF34, and provided a potential drug target for the control of NNV infection. Author Summary Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a kind of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. Previous study reported NNV could hijack the ubiquitin system to manipulate the host’s immune responses, however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase RNF34 as an IFN antagonist to promote NNV infection. Nervous necrosis virus capsid protein utilized RNF34 to target TBK1 and IRF3 for K27 and K48-linked ubiquitination degradation. Importantly, the interactions between RNF34 and CP, TBK1 or IRF3 are conserved in different fishes, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

Nervous Necrosis Virus Capsid Protein Functions as a SUMO E3 Ligase to Activate MAVS-Dependent NF-κB Signaling

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