USP13 Cooperates with MARCH8 to Inhibit Antiviral Signaling by Targeting MAVS for Autophagic Degradation in Teleost.

Mitochondrial antiviral signaling protein (MAVS), as a central adapter protein in retinoic acid-inducible gene I-like receptor signaling, is indispensable for innate antiviral immunity. Yet, the molecular mechanisms modulating the stability of MAVS are not fully understood in low vertebrates. In this study, we report that the deubiquitinase ubiquitin-specific protease 13 (USP13) acts as a negative regulator of antiviral immunity by targeting MAVS for selective autophagic degradation in teleost fish. USP13 is induced by RNA virus or polyinosinic:polycytidylic acid stimulation and acts as a negative regulator to potentiate viral replication in fish cells. Mechanistically, USP13 functions as a scaffold to enhance the interaction between MAVS and the E3 ubiquitin ligase MARCH8, thus promoting MARCH8 to catalyze MAVS through K27-linked polyubiquitination for selective autophagic degradation. Taken together, to our knowledge, our study demonstrates a novel mechanism by which viruses evade host antiviral immunity via USP13 in fish and provides a new idea for mammalian innate antiviral immunity.

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

circMORC3-encoded novel protein negatively regulates antiviral immunity through synergizing with host gene MORC3.

The protein-coding ability of circRNAs has recently been a hot topic, but the role of protein-coding circRNAs in antiviral innate immunity of teleost fish has rarely been reported. Here, we identified a novel circRNA, termed circMORC3, derived from Microrchidia 3 (MORC3) gene in Miichthys miiuy. circMORC3 can inhibit the expression of antiviral cytokines. In addition, circMORC3 encodes a novel peptide with a length of 84 amino acids termed MORC3-84aa. MORC3-84aa not only significantly inhibited TRIF-mediated activation of IRF3 and NF-κB signaling pathways, but also effectively suppressed the expression of antiviral cytokines triggered by RNA virus Siniperca chuatsi rhabdovirus (SCRV). We found that MORC3-84aa directly interacted with TRIF and negatively regulated TRIF protein expression. In addition, host gene MORC3 attenuates SCRV-induced IFN and ISG expression. Mechanistically, MORC3-84aa promotes autophagic degradation of TRIF by enhancing K6-linked ubiquitination and inhibits TRIF-mediated activation of the type I interferon signaling pathway. And the host gene MORC3 not only repressed IRF3 protein expression but also inhibited IRF3 phosphorylation levels. Our study shows that circMORC3 and host gene MORC3 played a synergistic role in viral immune escape.

METTL3-Mediated m6A Modification of TRIF and MyD88 mRNAs Suppresses Innate Immunity in Teleost Fish, Miichthys miiuy.

Methyltransferase (METTL3), the most important N6-methyladenosine (m6A) writer, plays a vital role in regulating immune-related signaling pathways. However, the underlying mechanism of METTL3 action remains largely unknown, especially in lower vertebrates. The results of this study show that METTL3 inhibits innate immune response and promotes the infection of miiuy croaker, Miichthys miiuy, by Siniperca chuatsi rhabdovirus and Vibrio anguillarum. Significantly, the function of METTL3 in inhibiting immunity depends on its methylase activity. Mechanistically, METTL3 increases the methylation level of trif and myd88 mRNA, rendering them sensitive to degradation by the YTHDF2/3 reader proteins. By contrast, we found that the YTHDF1 reader protein promotes the translation of myd88 mRNA. In summary, these results indicate that METTL3-mediated m6A modification of trif and myd88 mRNAs suppresses innate immunity by inhibiting the TLR pathway, unveiling a molecular mechanism by which RNA methylation controls innate immunity to pathogens in the teleost fish.

The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.

At present, N6-methyladenosine (m6A) modification has been proven to participate in a wide range of gene expression regulation, such as stability, translation, splicing, and output, among others, which has attracted much attention. Unlike mammals, however, the role of m6A in innate immunity of lower invertebrates has not yet been studied. In this study, we found that the total m6A level of Miichthys miiuy increased during Siniperca chuatsi rhabdovirus and Vibrio anguillarum infection, suggesting that m6A may play an important role in the immune process against pathogens in fish. In addition, our study shows that stimulator of IFN genes (STING) plays a dual immune function against viruses and bacteria in fish, and through degrading STING by identifying its m6A methylation site modified by methyltransferase-like 3 (METTL3), YTH domain family 2 (YTHDF2) can weaken the IRF3 and NF-κB-driven signaling pathway, thus weakening the innate immunity and promoting the infection of Siniperca chuatsi rhabdovirus and V. anguillarum to the M. miiuy. Although there have been reports on m6A modification of STING in mammals, it is still unclear whether there is also m6A modification in lower vertebrates, especially in fish. Therefore, our study provides a reference for filling the gap of m6A modification between fish and mammals.

CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape.

It is known that about 10 circular RNAs (circRNAs) can encode functional polypeptides in higher mammals. However, it is not clear whether the functional polypeptides that can be translated by circRNAs are only the products of the evolution of higher animals, or also widely exist in other lower organisms. In addition, it is also unclear whether the two ways of translating polypeptides using IRES and m6A in the one circRNA are exclusive or coexistent. Here, we discovered a novel circRNA derived from the 3'-5' RNA helicase Ythdc2 (Ythdc2) gene in lower vertebrate fish, namely circYthdc2, which can translate into a 170 amino acid polypeptide (Ythdc2-170aa) through IRES sequence or m6A modification, and is involved in antiviral immune of fish. Moreover, SCRV infection can promote circYthdc2 translate Ythdc2-170aa. Then, we found that both Ythdc2-170aa and Ythdc2 can promote the degradation of STING by promoting the ubiquitination modification of K11 and K48 link of STING, and weaken the host's antiviral innate immunity. Notably, when circYthdc2 is abundant, Ythdc2 preferentially degrades circYthdc2 and no longer promotes the degradation of STING. Further studies have shown that circYthdc2 is highly conserved from lower vertebrates to higher mammals, and human circYthdc2 can also encode the same polypeptide and play a similar function to that of fish circYthdc2. This discovery confirms for the first time that the ability of circRNA to encode functional proteins is evolutionarily conserved, and finds that the ways of polypeptide translation by the same circRNA were diverse, which is of great significance for further elucidating the function and evolution of circRNAs in vertebrates.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

IMPORTANCE: The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

Smyd3 negatively regulates the anti-viral pathway by promoting TAK1 degradation in teleost fish.

IMPORTANCE: In this study, we have found that the existence of Smyd3 promoted the replication of SCRV. Additionally, we report that Smyd3 negatively regulates the NF-κB and IRF3 signaling pathway by facilitating the degradation of TAK1 in fish. Our findings suggest that Smyd3 interacts with TAK1. Further investigations have revealed that Smyd3 specifically mediates K48-linked ubiquitination of TAK1 and enhances TAK1 degradation, resulting in a significant inhibition of the NF-κB and IRF3 signaling pathway. These results not only contribute to the advancement of fish anti-viral immunity but also provide new evidence for understanding the mechanism of TAK1 in mammals.

circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish.

IMPORTANCE: Increasing evidence indicates that circular RNAs exert crucial functions in regulating gene expression in mammals. However, the function of circRNAs in lower vertebrates still needs further exploration. Our research results demonstrated that circRNA, namely circCBL, is involved in modulating antiviral and antibacterial immune responses in lower vertebrates. In addition, our study also found that circCBL can serve as a competing endogenous RNA to facilitate MITA expression, thereby modulating MITA-mediated innate immunity. Further research has proved that the host gene CBL also promotes the expression of MITA, enhancing antiviral and antibacterial immune responses. Our study not only elucidated the underlying biological mechanism of the circRNA-miRNA-mRNA axis in the innate immune response of lower vertebrates but also unveiled the synergistic antibacterial and antiviral mechanisms between circRNA and its host gene in lower vertebrates.

STAM2 negatively regulates the MyD88-mediated NF-κB signaling pathway in miiuy croaker, Miichthys miiuy.

Signal transducing adapter molecule 2 (STAM2), a member of the Signal Transducing Adapter Molecule (STAM) family, is a protein with significant implications in diverse signaling pathways and endocytic membrane trafficking. However, the role of the STAM2, especially in fish, remains largely unknown. In this study, we discovered that STAM2 negatively regulates the NF-κB signaling pathway, and its inhibitory effect is enhanced upon LPS induction. Our study confirmed that STAM2 can enhance the degradation of myeloid differentiation primary-response protein 88 (MyD88), an upstream regulator of NF-κB pathway. Furthermore, the UIM domain of STAM2 is important for the inhibition of MyD88. Mechanistically, STAM2 inhibits the NF-κB signaling pathway by targeting the MyD88 autophagy pathway. In addition, we showed that STAM2 promotes the proliferation of Vibrio harveyi. In summary, our study reveals that STAM2 inhibits NF-κB signaling activation and mediates innate immunity in teleost via the autophagy pathway.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.

The long noncoding RNA MIR122HG is a precursor for miR-122-5p and negatively regulates the TAK1-induced innate immune response in teleost fish.

Long noncoding RNAs (lncRNAs) are a diverse subset of RNA species of noncoding transcripts that are usually longer than 200 nt. However, the biological role and function of many lncRNAs have not been fully identified. It has been shown that one potential function of lncRNAs is to act as a precursor miRNA and promote the production of multiple miRNAs. However, the function of the miiuy croaker lncRNA MIR122HG has not been explored. In the present study, we show that this differentially expressed teleost fish lncRNA can act as the host gene of miR-122-5p, regulate its expression, and indirectly regulate the expression of potential inflammatory target protein transforming growth factor-ß-activated kinase 1. We show that MIR122HG can negatively regulate the transforming growth factor-ß-activated kinase 1-triggered NF-κB and interferon regulatory factor 3 signaling pathways and subsequently attenuate the innate immune response. In addition, MIR122HG can promote the replication of Siniperca chuatsi rhabdovirus and exacerbate the pathological effects caused by viral infection. We conclude that the study of lncRNA-miRNA-mRNA interaction through bioinformatics analysis or experimental-supported analysis can provide information for further elucidation of the functions of fish lncRNAs in innate immunity.

eIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish.

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host's autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.

Long Noncoding RNA MIR2187HG Suppresses TBK1-Mediated Antiviral Signaling by Deriving miR-2187-3p in Teleost Fish.

Long noncoding RNAs (lncRNAs) function as microregulatory factors that influence gene expression after a variety of pathogenic infections, and they have been extensively studied in the past few years. Although less attention has been paid to lncRNAs in lower vertebrates than in mammals, current studies reveals that lncRNAs play a vital role in fish stimulated by pathogens. Here, we discovered a new lncRNA, termed MIR2187HG, which can function as a precursor of a small RNA, miR-2187-3p, with regulatory functions in the miiuy croaker (Miichthys miiuy). Upon Siniperca chuatsi rhabdovirus (SCRV) virus infection, the expression levels of MIR2187HG were remarkably enhanced. Elevated MIR2187HG expression can act as a pivotally negative regulator that participates in the innate immune response of teleost fish to inhibit the intracellular TANK-binding kinase 1 (TBK1)-mediated antiviral signaling pathways, which can effectively avoid excessive immunity. In addition, we found that SCRV could also utilize MIR2187HG to enhance its own numbers. Our results not only provide evidence regarding the involvement of the lncRNAs in response to viruses in fish but also broaden our understanding of the function of lncRNAs as precursor microRNAs (miRNAs) in teleost fish for the first time. IMPORTANCE SCRV infection upregulates MIR2187HG levels, which in turn suppresses SCRV-triggered type I interferon production, thus promoting viral replication in miiuy croaker. Notably, MIR2187HG regulates the release of miR-2187-3p, and TBK1 is a target of miR-2187-3p. MIR2187HG could acquire from miR-2187-3p the function of inhibiting TBK1 expression and subsequently modulate TBK1-mediated NF-κB and IRF3 signaling. The collective results suggest that the novel regulation mechanism of TBK1-mediated antiviral response during RNA viral infection was regulated by MIR2187HG. Therefore, a new regulation mechanism for lncRNAs to regulate antiviral immune responses in fish is proposed.

Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish.

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that exert crucial functions in regulating gene expression in mammals. However, the function and regulation mechanism of circRNAs in lower vertebrates are still unknown. Here, we discovered a novel circRNA derived from Deltex E3 ubiquitin ligase 1 (Dtx1) gene, namely, circDtx1, which was related to the antiviral responses in teleost fish. Results indicated that circDtx1 played essential roles in host antiviral immunity and inhibition of SCRV replication. Our study also found a microRNA miR-15a-5p, which could inhibit antiviral immune response and promote viral replication by targeting TRIF. Moreover, we also found that the antiviral effect inhibited by miR-15a-5p could be reversed with the circDtx1. In mechanism, our data revealed that circDtx1 was a competing endogenous RNA (ceRNA) of TRIF by sponging miR-15a-5p, leading to activation of the NF-κB/IRF3 pathway, and then enhancing the innate antiviral responses. Our results indicated that circRNAs played a regulatory role in immune responses in teleost fish.

PSMD13 inhibits NF-κB pathway by targeting TAK1 for K63-linked ubiquitination in miiuy croaker (Miichthys miiuy).

Transforming growth factor-ß activated kinase 1 (TAK1) is an adaptor molecular in the TLR-mediated NF-κB pathway which has been implicated in the regulation of a wide range of physiological and pathological processes. Proteasome 26S subunit, non-ATPase (PSMD) 13 is essential for the structural maintenance and function of the 26S proteasome. However, the mechanism of PSMD13 in innate immune regulation is not clear. In this study, the expression of PSMD13 mRNA was significantly increased under Vibrio harveyi stimulation, and PSMD13 inhibited the NF-κB pathway by targeting TAK1. Mechanically, PSMD13 significantly inhibited the K63-linked ubiquitination of TAK1, thereby inhibiting the expression of TAK1. Moreover, this discovery enriches the research of the PSMD family in regulating the innate immune response and provides a new idea for the study of the mammalian innate immune regulation mechanism.

Vinculin B inhibits NF-κB signaling pathway by targeting MyD88 in miiuy croaker, Miichthys miiuy.

Myeloid differentiation factor 88 (MyD88) is the canonical adaptor for inflammatory signaling pathways downstream from members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families, which activates the NF-κB signaling pathway and regulates immune and inflammatory responses. In this study, we found that Vinculin B (Vclb) is an inhibitor in the NF-κB signaling pathway, and its inhibitory effect was enhanced by LPS induction. Furthermore, Vclb inhibits NF-κB activation by targeting MyD88, thereby suppressing the production of inflammatory cytokines. Mechanistically, Vclb inhibits the NF-κB signaling pathway by targeting MyD88 ubiquitin-proteasome pathway. In summary, our study reveals that Vclb inhibits NF-κB signaling activation and mediates innate immunity in teleosts via the ubiquitin-proteasome pathway of MyD88.

Identification of a novel fusion gene NLRC3-NLRP12 in miiuy croaker (Miichthys miiuy).

Fusion gene is a new gene formed by the fusion of all or part of the sequences of two genes, it is caused by chromosome translocation, middle deletion or chromosome inversion. Numerous studies in the past have continuously shown that gene fusions are tightly associated with the occurrence and development of various diseases, especially cancer. Many fusion genes have been identified in humans. However, few fusion genes have been identified in fish. In this study, a novel NLRC3-NLRP12 fusion gene was identified in the Miichthys miiuy (miiuy croaker) by quantitative real-time PCR (qRT-PCR), PCR, and Sanger sequencing. This fusion gene is fused by two genes related to NLRs (nucleotide binding domain and oligomerization domain like receptors). We found that the expression of the NLRC3-NLRP12 fusion gene was significantly upregulated after infection with Vibrio anguillarum (V. anguillarum) or stimulation with lipopolysaccharide (LPS). In addition, the NLRC3-NLRP12 fusion gene was strongly induced by V. anguillarum infection, peaking within the kidney and liver at 12 h post infection. Further functional experiments showed that overexpression of NLRC3-NLRP12 significantly inhibited nuclear factor kappa-B (NF-κB) activation. This study suggests that the newly discovered NLRC3-NLRP12 fusion genes may play an important role in innate immunity in miiuy croaker.

Circular RNA circPlce1 regulates innate immune response in miiuy croaker, Miichthys miiuy.

In recent years, more and more researchers have devoted to the study of circular RNAs (circRNAs) in noncoding RNAs. As an important regulator in a variety of biological processes, circRNAs are relatively abundant in the study of mammals, while research in lower vertebrates is still lacking. In this study, we found a circRNA, circPlce1, related to innate immune response in Miichthys miiuy (miiuy croaker). The experimental results confirmed that circPlce1 could promote the production of antiviral genes and inflammatory response under the stimulation of poly (I: C) and LPS. We also confirmed that circPlce1 can promote NF-κB and IRF3 pathways through luciferase reporter assay experiment. In addition, we also found that circPlce1 can promote cell proliferation and improve cell viability. In conclusion, our results showed that circPlce1 plays an active role in regulating inflammatory response, cell proliferation and cell viability, providing a foundation for the study of the biological function of circRNAs in the innate immune response in teleost fish.