Zebrafish as a Model for Investigating Antiviral Innate Immunity.

Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model.

Coordination of oxysterol binding protein 1 and VAP-A/B modulates the generation of cholesterol and viral inclusion bodies to promote grass carp reovirus replication.

Similar to other RNA viruses, grass carp reovirus, the causative agent of the hemorrhagic disease, replicates in cytoplasmic viral inclusion bodies (VIBs), orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of GCRV VIBs are poorly understood. This work demonstrates that GCRV manipulates grass carp oxysterol binding protein 1 (named as gcOSBP1) and vesicle-associated membrane protein-associated protein A/B (named as gcVAP-A/B), 3 components of cholesterol transport pathway, to generate VIBs. By siRNA-mediated knockdown, we demonstrate that gcOSBP1 is an essential host factor for GCRV replication. We reveal that the nonstructural proteins NS80 and NS38 of GCRV interact with gcOSBP1, and that the gcOSBP1 is recruited by NS38 and NS80 for promoting the generation of VIBs. gcOSBP1 increases the expression of gcVAP-A/B and promotes the accumulation of intracellular cholesterol. gcOSBP1 also interacts with gcVAP-A/B for forming gcOSBP1-gcVAP-A/B complexes, which contribute to enhance the accumulation of intracellular cholesterol and gcOSBP1-mediated generation of VIBs. Inhibiting cholesterol accumulation by lovastatin can completely abolish the effects of gcOSBP1 and/or gcVAP-A/B in promoting GCRV infection, suggesting that cholesterol accumulation is vital for gcOSBP1- and/or gcVAP-A/B-mediated GCRV replication. Thus, our results, which highlight that gcOSBP1 functions in the replication of GCRV via its interaction with essential viral proteins for forming VIBs and with host gcVAP-A/B, provide key molecular targets for obtaining anti-hemorrhagic disease grass carp via gene editing technology.

The dexd/H-box helicase DHX40 is a novel negative regulator during GCRV infection via targeting the host RLR helicases and viral nonstructural protein NS38.

RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.

Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

Advances in Bacterial Oligosaccharyltransferase Structure Elucidation and Potential Application to Glycoconjugate Vaccine Design.

Glycosylation is one of the most common post-translational modifications of proteins across all kingdoms of life. Diverse monosaccharides and polysaccharides can be attached to a range of amino acid residues generating N-glycosylation, O-glycosylation, C-glycosylation, S-glycosylation, as well as P-glycosylation. The functions of the eukaryotic glycosylation system during protein folding in the endoplasmic reticulum (ER) and Golgi are well-studied. Increasing evidence in the recent decade has demonstrated the presence of oligosaccharyltransferases (OSTs) in bacteria and archaea. In particular, the oligosaccharyltransferase (PglB) of Campylobacter jejuni and oligosaccharyltransferase (PglL) enzyme of Neisseria meningitidis are the most characterized OSTs that catalyze bacterial N-linked glycosylation and O-linked glycosylation, respectively. Glycoprotein administered as glycoconjugate vaccines have been shown to be effective prophylactic to protect against numerous pathogenic bacteria. The chemical synthesis of glycoproteins is complex and expensive, which limits its application to the development of glycoconjugate vaccines. However, studies have demonstrated that the biosynthesis of glycoproteins is realizable by transferring PglB, a plasmid encoding a substrate protein, or PglL, a plasmid encoding genes for glycan synthesis to Escherichia coli. This strategy can be applied to the development of glycoconjugate vaccines using engineered host E. coli. This review summarizes the structure and mechanism of action of the bacterial OSTs, PglB and PglL, and discusses their potential application to glycoconjugate vaccine design.

Zebrafish nos2a benefits bacterial proliferation via suppressing ROS and inducing NO production to impair the expressions of inflammatory cytokines and antibacterial genes.

The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.

Liver X Receptor LXRα Promotes Grass Carp Reovirus Infection by Attenuating IRF3-CBP Interaction and Inhibiting RLR Antiviral Signaling.

Liver X receptors (LXRs) are nuclear receptors involved in metabolism and the immune response. Different from mammalian LXRs, which include two isoforms, LXRα and LXRß, only a single LXRα gene exists in the piscine genomes. Although a study has suggested that piscine LXR inhibits intracellular bacterial survival, the functions of piscine LXRα in viral infection are unknown. In this study, we show that overexpression of LXRα from grass carp (Ctenopharyngodon idellus), which is named as gcLXRα, increases host susceptibility to grass carp reovirus (GCRV) infection, whereas gcLXRα knockdown in CIK (C. idellus kidney) cells inhibits GCRV infection. Consistent with these functional studies, gcLXRα knockdown promotes the transcription of antiviral genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway, including IFN regulatory factor (IRF3) and the type I IFN IFN1. Further results show that gcLXRα knockdown induces the expression of CREB-binding protein (CBP), a transcriptional coactivator. In the knockdown of CBP, the inhibitory effect of gcLXRα knockdown in limiting GCRV infection is completely abolished. gcLXRα also interacts with IRF3 and CBP, which impairs the formation of the IRF3/CBP transcription complex. Moreover, gcLXRα heterodimerizes with RXRg, which cooperatively impair the transcription of the RLR antiviral signaling pathway and promote GCRV infection. Taken together, to our knowledge, our findings provide new insight into the functional correlation between nuclear receptor LXRα and the RLR antiviral signaling pathway, and they demonstrate that gcLXRα can impair the RLR antiviral signaling pathway and the production of type I IFN via forming gcLXRα/RXRg complexes and attenuating IRF3/CBP complexes.

Piscine Vitamin D Receptors Vdra/Vdrb in the Absence of Vitamin D Are Utilized by Grass Carp Reovirus for Promoting Viral Replication.

The vitamin D receptor (VDR) plays a pivotal role in the biological actions of vitamin D (VitD). However, little is known about the functions of VDR in the production of viral inclusion bodies (VIBs). Using a representative strain of grass carp reovirus (GCRV) genotype I, GCRV-873, we show that GCRV-873 recruits grass carp Vdrs for promoting the production of VIBs in the absence of VitD. Inhibition of cholesterol synthesis by lovastatin impairs the production of VIBs and blocks the effects of grass carp Vdrs in promoting the production of VIBs in the absence of VitD. Furthermore, grass carp Vdrs are found to form the Vdra-Vdrb heterodimer, which is vital for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgcr)-dependent cholesterol synthesis and GCRV replication. Intriguingly in the presence of VitD, grass carp Vdra but not Vdrb forms the heterodimer with the retinoid X receptor beta b (Rxrbb), which induces the transcription of those genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway for inhibiting GCRV infection. Furthermore, the VitD-activated Vdra-Vdrb heterodimer attenuates the transcription of the RLR antiviral signaling pathway induced by VitD. In the presence of VitD, a balance between the Vdra-Rxrbb heterodimers as coactivators and Vdra-Vdrb heterodimers as corepressors in affecting the transcriptional regulation of the RLR antiviral signaling pathway may eventually determine the outcome of GCRV infection. Transfection with VitD can abolish the effect of grass carp Vdrs in promoting GCRV replication in a dose-dependent manner. Taken together, these findings demonstrate that GCRV utilizes host Vdrs to increase hmgcr-dependent cholesterol synthesis for promoting its replication, which can be prevented by VitD treatment. IMPORTANCE Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhagic disease, which seriously harms freshwater fish. Although many positive or negative regulators of GCRV infection have been identified in teleosts, little is known about the molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection. Here, we show that in the absence of VitD, the GCRV-873 strain utilizes host vitamin D receptors Vdra/Vdrb to increase hmgcr-dependent cholesterol synthesis for promoting the production of VIBs, which are important functional sites for aquareovirus replication and assembly. The negative regulation of Vdrs during viral infection can be prevented by VitD treatment. Thus, this present work broadens understanding of the pivotal roles of Vdrs in the interaction between the host and GCRV in the absence or presence of VitD, which might provide a rational basis for developing novel anti-GCRV strategies.

Emerging mechanisms and functions of inflammasome complexes in teleost fish.

Inflammasomes are multiprotein complexes, which are assembled in response to a diverse range of exogenous pathogens and endogenous danger signals, leading to produce pro-inflammatory cytokines and induce pyroptotic cell death. Inflammasome components have been identified in teleost fish. Previous reviews have highlighted the conservation of inflammasome components in evolution, inflammasome function in zebrafish infectious and non-infectious models, and the mechanism that induce pyroptosis in fish. The activation of inflammasome involves the canonical and noncanonical pathways, which can play critical roles in the control of various inflammatory and metabolic diseases. The canonical inflammasomes activate caspase-1, and their signaling is initiated by cytosolic pattern recognition receptors. However the noncanonical inflammasomes activate inflammatory caspase upon sensing of cytosolic lipopolysaccharide from Gram-negative bacteria. In this review, we summarize the mechanisms of activation of canonical and noncanonical inflammasomes in teleost fish, with a particular focus on inflammasome complexes in response to bacterial infection. Furthermore, the functions of inflammasome-associated effectors, specific regulatory mechanisms of teleost inflammasomes and functional roles of inflammasomes in innate immune responses are also reviewed. The knowledge of inflammasome activation and pathogen clearance in teleost fish will shed new light on new molecular targets for treatment of inflammatory and infectious diseases.

TBK1 Isoform Inhibits Grass Carp Reovirus Infection by Targeting the Degradation of Viral Nonstructural Proteins NS80 and NS38.

TANK-binding kinase 1 (TBK1) undergoes alternative splicing, and the previously reported TBK1 isoforms are negative regulators of RIG-I-like receptor-mediated type I IFN production. Although a study has suggested that grass carp TBK1 has an opposite effect at high- and low-titer of grass carp reovirus (GCRV) infection, the functions of grass carp TBK1 isoforms in GCRV infection remain unclear. In this study, we show that a TBK1 isoform from grass carp (Ctenopharyngodon idellus) named as gcTBK1_tv3, which has a 1-aa difference with zebrafish TBK1_tv3, inhibits the replication and infection of GCRV both at high and low titers of infection in C. idellus kidney cells. gcTBK1_tv3 can colocalize and interact with the NS80 and NS38 proteins of GCRV. Furthermore, gcTBK1_tv3 specifically degrades the NS80 and NS38 proteins of GCRV through the ubiquitin-proteasome pathway. Mechanistically, gcTBK1_tv3 promotes the degradation of NS80 or NS38 for K48-linked ubiquitination by targeting the Lys503 residue of NS80 or Lys328 residue of NS38, respectively, which ultimately impairs the production of cytoplasmic viral inclusion bodies and limits GCRV replication and infection. Taken together, our findings provide insight into the function of TBK1 isoform in the antiviral immune response and demonstrate that TBK1 isoform can target the nonstructural proteins of GCRV for impairing the formation of viral inclusion bodies.

Structural and functional analysis of the small GTPase ARF1 reveals a pivotal role of its GTP-binding domain in controlling of the generation of viral inclusion bodies and replication of grass carp reovirus.

Grass carp reovirus (GCRV) is the most pathogenic double-stranded (ds) RNA virus among the isolated aquareoviruses. The molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection are poorly understood. Here, we discovered that the grass carp ADP ribosylation factor 1 (gcARF1) was required for GCRV replication since the knockdown of gcARF1 by siRNA or inhibiting its GTPase activity by treatment with brefeldin A (BFA) significantly impaired the yield of infectious viral progeny. GCRV infection recruited gcARF1 into viral inclusion bodies (VIBs) by its nonstructural proteins NS80 and NS38. The small_GTP domain of gcARF1 was confirmed to be crucial for promoting GCRV replication and infection, and the number of VIBs reduced significantly by the inhibition of gcARF1 GTPase activity. The analysis of gcARF1-GDP complex crystal structure revealed that the 27AAGKTT32 motif and eight amino acid residues (A27, G29, K30, T31, T32, N126, D129 and A160), which were located mainly within the GTP-binding domain of gcARF1, were crucial for the binding of gcARF1 with GDP. Furthermore, the 27AAGKTT32 motif and the amino acid residue T31 of gcARF1 were indispensable for the function of gcARF1 in promoting GCRV replication and infection. Taken together, it is demonstrated that the GTPase activity of gcARF1 is required for efficient replication of GCRV and that host GTPase ARF1 is closely related with the generation of VIBs.

CD44a functions as a regulator of p53 signaling, apoptosis and autophagy in the antibacterial immune response.

The cell adhesion molecule CD44 has been implicated in diverse biological functions including the pathological responses to infections and inflammatory diseases. The variable forms of CD44 contribute to functional variations, which are not yet defined in teleost. Here, we show that zebrafish CD44a plays a protective role in the host defense against Edwardsiella piscicida infection. Zebrafish CD44a deficiency inhibits cell growth and proliferation, impairs cell growth and death pathways, and regulates the expression levels of many genes involved in p53 signaling, apoptosis and autophagy. In addition, CD44a gene disruption in zebrafish leads to inhibition of apoptosis and induction of autophagy, with the increased susceptibility to E. piscicida infection. Furthermore, we show that zebrafish CD44a variants including CD44a_tv1 and CD44a_tv2 promote the translocation of p53 from the nucleus to the cytoplasm and interact with p53 in the cytoplasm. Mechanistically, zebrafish CD44a_tv1 mediates the beneficial effect for larvae survival infected with E. piscicida is depending on the CASP8-mediated apoptosis. However, the antibacterial effect of zebrafish CD44a_tv2 depends on the cytoplasmic p53-mediated inhibition of autophagy. Collectively, our results identify that different mechanisms regulate CD44a variants-mediated antibacterial responses.

Immunoprotective Effects of Two Histone H2A Variants in the Grass Carp Against Flavobacterium columnare Infection.

In teleost fish, the nucleotide polymorphisms of histone H2A significantly affect the resistance or susceptibility of zebrafish to Edwardsiella piscicida infection. Whether histone H2A variants can enhance the resistance of grass carp to Flavobacterium columnare infection remains unclear. Here, the effects of 7 previously obtained variants (gcH2A-1~gcH2A-7) and 5 novel histone H2A variants (gcH2A-11, gcH2A-13~gcH2A-16) in response to F. columnare infection were investigated. It was found that these histone H2A variants could be divided into type I and II. Among them, 5 histone H2A variants had no any effects on the F. columnare infection, however 7 histone H2A variants had antibacterial activity against F. columnare infection. The gcH2A-4 and gcH2A-11, whose antibacterial activity was the strongest in type I and II histone H2A variants respectively, were picked out for yeast expression. Transcriptome data for the samples from the intestines of grass carp immunized with the engineered Saccharomyces cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 revealed that 5 and 12 immune-related signaling pathways were significantly enriched by gcH2A-4 or gcH2A-11, respectively. For the engineered S. cerevisiae expressing gcH2A-4, NOD-like receptor and Toll-like receptor signaling pathways were enriched for up-regulated DEGs. Besides NOD-like receptor and Toll-like receptor signaling pathways, the engineered S. cerevisiae expressing gcH2A-11 also activated Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway and C-type lectin receptor signaling pathway. Furthermore, grass carp were immunized with the engineered S. cerevisiae expressing PYD1, gcH2A-4 or gcH2A-11 for 1 month and challenged with F. columnare. These grass carp immunized with gcH2A-4 or gcH2A-11 showed lower mortality and fewer numbers of F. columnare than did the control group. All these results suggest that gcH2A-4 and gcH2A-11 play important roles in evoking the innate immune responses and enhancing disease resistance of grass carp against F. columnare infection.

Effects and Molecular Regulation Mechanisms of Salinity Stress on the Health and Disease Resistance of Grass Carp.

Though some freshwater fish have been successfully cultivated in saline-alkali water, the survival rates of freshwater fish are greatly affected by different saline-alkali conditions. The mechanisms of immune adaptation or immunosuppression of freshwater fish under different saline-alkali stress remain unclear. Here, grass carp were exposed to 3‰ and 6‰ salinity for 30 days. It was observed that salinity treatments had no obvious effects on survival rates, but significantly increased the percent of unhealthy fish. Salinity treatments also increased the susceptibility of grass carp against Flavobacterium columnare infection. The fatality rate (16.67%) of grass carp treated with 6‰ salinity was much lower than that treated with 3‰ salinity (40%). In the absence of infection, higher numbers of immune-related DEGs and signaling pathways were enriched in 6‰ salinity-treated asymptomatic fish than in 3‰ salinity-treated asymptomatic fish. Furthermore different from salinity-treated symptomatic fish, more DEGs involved in the upstream sensors of NOD-like receptor signaling pathway, such as NLRs, were induced in the gills of 6‰ salinity-treated asymptomatic fish. However in the case of F. columnare infection, more immune-related signaling pathways were impaired by salinity treatments. Among them, only NOD-like receptor signaling pathway was significantly enriched at early (1 and/or 2 dpi) and late (7 dpi) time points of infection both for 3‰ salinity-treated and 6‰ salinity-treated fish. Besides the innate immune responses, the adaptive immune responses such as the production of Ig levels were impaired by salinity treatments in the grass carp infected with F. columnare. The present study also characterized two novel NLRs regulated by salinity stress could inhibit bacterial proliferation and improve the survival rate of infected cells. Collectively, the present study provides the insights into the possible mechanisms why the percent of unhealthy fish in the absence of infection and mortality of grass carp in the case of F. columnare infection were much lower in the 6‰ salinity-treated grass carp than in 3‰ salinity-treated grass carp, and also offers a number of potential markers for sensing both environmental salinity stress and pathogen.

Grass Carp Reovirus Nonstructural Proteins Avoid Host Antiviral Immune Response by Targeting the RLR Signaling Pathway.

Grass carp reovirus (GCRV) is a highly virulent RNA virus that mainly infects grass carp and causes hemorrhagic disease. The roles of nonstructural proteins NS38 and NS80 of GCRV-873 in the viral replication cycle and viral inclusion bodies have been established. However, the strategies that NS38 and NS80 used to avoid host antiviral immune response are still unknown. In this study, we report the negative regulations of NS38 and NS80 on the RIG-I-like receptors (RLRs) antiviral signaling pathway and the production of IFNs and IFN-stimulated genes. First, both in the case of overexpression and GCRV infection, NS38 and NS80 inhibited the IFN promoter activation induced by RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7 and mRNA abundance of key antiviral genes involved in the RLR-mediated signaling. Second, both in the case of overexpression and GCRV infection, NS38 interacted with piscine TBK1 and IRF3, but not with piscine RIG-I, MDA5, MAVS, and TNF receptor-associated factor (TRAF) 3. Whereas NS80 interacted with piscine MAVS, TRAF3, and TBK1, but not with piscine RIG-I, MDA5, and IRF3. Finally, both in the case of overexpression and GCRV infection, NS38 inhibited the formation of the TBK1-IRF3 complex, but NS80 inhibited the formation of the TBK1-TRAF3 complex. Most importantly, NS38 and NS80 could hijack piscine TBK1 and IRF3 into the cytoplasmic viral inclusion bodies and inhibit the translocation of IRF3 into the nucleus. Collectively, all of these data demonstrate that GCRV nonstructural proteins can avoid host antiviral immune response by targeting the RLR signaling pathway, which prevents IFN-stimulated gene production and facilitates GCRV replication.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

The Zebrafish Antiapoptotic Protein BIRC2 Promotes Edwardsiella piscicida Infection by Inhibiting Caspases and Accumulating p53 in a p53 Transcription-Dependent and -Independent Manner.

IAPs (inhibitors of apoptosis) are endogenous caspase inhibitors with multiple biological activities. In the present study, we show functional characteristics of antiapoptotic protein BIRC2 (cIAP1) in response to Edwardsiella piscicida infection. Overexpression of BIRC2 in zebrafish larvae promoted the proliferation of E. piscicida, leading to a decreased larvae survival. The expression levels of caspases including casp3, casp8, and casp9 were significantly inhibited by BIRC2 overexpression in the case of E. piscicida infection. Treatment of zebrafish larvae microinjected with BIRC2 with the caspase activator PAC-1 completely blocked the negative regulation of BIRC2 on the E. piscicida infection, with the reduced inhibition on the casp3 and without inhibition on casp8 and casp9. In contrast to the regulation of BIRC2 on the caspases, BIRC2 overexpression significantly induced the expression of p53, especially at 24 hpi. In addition to the cytoplasmic p53 expression, BIRC2 overexpression also induced the expression of the nuclear p53 protein. Further analysis demonstrated that BIRC2 could interact and colocalize with p53 in the cytoplasm. The numbers of E. piscicida in larvae overexpressed with BIRC2 and treated with pifithrin-µ (an inhibitor of mitochondrial p53) or pifithrin-α (an inhibitor of p53 transactivation) were lower than those of larvae without pifithrin-µ or pifithrin-α treatment. Critically, the p53 inactivators pifithrin-µ and pifithrin-α had no significant effect on larval survival, but completely rescued larval survival for zebrafish microinjected with BIRC2 in the case of E. piscicida infection. Collectively, the present study suggest that piscine BIRC2 is a negative regulator for antibacterial immune response in response to the E. piscicida infection via inhibiting caspases, and accumulating p53 in a p53 transcription-dependent and -independent manner.

The negative regulation of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway in fish.

At each stage of innate immune response, there are stimulatory and inhibitory signals that modulate the strength and character of the response. RIG-I-like receptor (RLR) signaling pathway plays pivotal roles in antiviral innate immune response. Recent studies have revealed the molecular mechanisms that viral infection leads to the activation of RLRs-mediated downstream signaling cascades and the production of type I interferons (IFNs). However, antiviral immune responses must be tightly regulated in order to prevent detrimental type I IFNs production. Previous reviews have highlighted negative regulation of RLR signaling pathway, which mainly target to directly regulate RIG-I, MDA5, MAVS and TBK1 function in mammals. In this review, we summarize recent advances in our understanding of negative regulators of RLR signaling pathway in teleost, with specific focus on piscine and viral regulatory mechanisms that directly or indirectly inhibit the function of RIG-I, MDA5, LGP2, MAVS, TRAF3, TBK1, IRF3 and IRF7 both in the steady state or upon viral infection. We also further discuss important directions for future studies, especially for non-coding RNAs and post-translational modifications via fish specific TRIM proteins. The knowledge of negative regulators of RLR signaling pathway in teleost will shed new light on the critical information for potential therapeutic purposes.

The expanding and function of NLRC3 or NLRC3-like in teleost fish: Recent advances and novel insights.

The nucleotide-binding domain and leucine-rich repeat-containing family (NLR) proteins are innate immune sensors which recognize highly conserved pathogen-associated molecular patterns (PAMPs). Mammals have small numbers of NLR proteins, whereas in some species such as in invertebrates and jawless vertebrates, NLRs have expanded into very large families. Nearly 400 NLR proteins are identified in the zebrafish genome. Members of the NLR family can be divided into two functional sub-groups based on their ability to either positively or negatively regulate host immune response or inflammatory signaling cascades. Mammalian NLRC3 has been identified as an inhibitory NLR, and serves as a negative regulator in the NF-κB-mediated inflammatory response, STING-mediated DNA sensing and PI3K-mTOR pathways. Different from mammalian NLRC3, the analysis from genomes or transcriptomes revealed that the expansions of NLRC3 existed in different species of fish. Furthermore, piscine NLRC3-like genes were confirmed to have a negative or positive regulatory function in response to different kinds of pathogen infections and in the production of proinflammatory cytokines. In this review, we summarize recent advances in our understanding of the expanding and function of NLRC3 or NLRC3-like genes in teleost fish, and give our view of important directions for future studies. The knowledge of piscine NLRC3 or expansive NLRC3-like genes-mediated biological functions in homeostasis and diseases will shed new light on the prevention and control of inflammatory and/or infectious diseases.

The function of zebrafish gpbar1 in antiviral response and lipid metabolism.

G protein-coupled bile acids receptor 1 (GPBAR1 or TGR5) has been widely studied as a metabolic regulator involved in bile acids synthesis, glucose metabolism and energy homeostasis. Several recent studies have shown that mammalian GPBAR1 is also involved in antiviral innate immune responses. However, the functions of piscine GPBAR1 in antibacterial or antiviral immune responses and lipid metabolism remain unclear. In the present study, we report the functional characterization of zebrafish gpbar1. Similar to mammalian GPBAR1, zebrafish gpbar1 contains similar domain composition, shows a dose-dependent activation by bile acids including INT777, LCA, DCA, CDCA and CA, and can be induced by viral infection. Compared with corresponding control groups, a significant antiviral activity against spring viremia of carp virus (SVCV) infection was observed in ZF4 cells overexpressing zebrafish gpbar1 with INT777 treatment, but not in ZF4 cells overexpressing zebrafish gpbar1 without INT777 treatment. The activation of zebrafish gpbar1 had no significant antibacterial effect against Edwardsiella piscicida infection in ZF4 cells in vitro. Transcriptome analysis revealed that zebrafish gpbar1 activation played a crucial role in activating RLR signaling pathway and inducing the production of ISGs, but not for bile acid biosynthesis and transportation. The co-occurrence analysis for antiviral-related and bile acids metabolism-related DEGs suggested a strong interaction among 2 bile acid receptors (gpbar1 and nr1h4), slco2b1 and the antiviral DEGs. The lipidomic analysis showed that zebrafish gpbar1 activation in ZF4 cells resulted a change of glycerophospholipids, but none of bile acids nor their derivatives, which were different from mammalian GPBAR1. All together, these results firstly demonstrate the conserved antiviral role of gpbar1 and its function in regulating glycerophospholipids metabolism in teleost.