RESUMEN
PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.
RESUMEN
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.
Asunto(s)
Carpas , Proteolisis , Receptores de Reconocimiento de Patrones , Rhabdoviridae , Proteínas de Motivos Tripartitos , Proteínas Virales , Proteínas de Pez Cebra , Pez Cebra , Animales , Carpas/virología , Proteína 58 DEAD Box/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/metabolismo , Inmunidad Innata , Receptores de Reconocimiento de Patrones/metabolismo , Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/virología , Transducción de Señal , Proteínas de Motivos Tripartitos/deficiencia , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitinación , Proteínas Virales/metabolismo , Viremia/veterinaria , Viremia/virología , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/virología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Spring viremia of carp virus (SVCV) is the causative agent of spring viremia of carp (SVC), an important infectious disease that causes high mortality in aquaculture cyprinids. How the host defends against SVCV infection and the underlying mechanisms are still elusive. In this study, we identify that a novel gene named maoc1 is induced by SVCV infection. maoc1-deficient zebrafish are more susceptible to SVCV infection, with higher virus replication and antiviral gene induction. Further assays indicate that maoc1 interacts with the P protein of SVCV to trigger P protein degradation through the autophagy-lysosomal pathway, leading to the restriction of SVCV propagation. These findings reveal a unique zebrafish defense machinery in response to SVCV infection. IMPORTANCE SVCV P protein plays an essential role in the virus replication and viral immune evasion process. Here, we identify maoc1 as a novel SVCV-inducible gene and demonstrate its antiviral capacity through attenuating SVCV replication, by directly binding to P protein and mediating its degradation via the autophagy-lysosomal pathway. Therefore, this study not only reveals an essential role of maoc1 in fighting against SVCV infection but also demonstrates an unusual host defense mechanism in response to invading viruses.
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Autofagia , Enfermedades de los Peces , Lisosomas , Infecciones por Rhabdoviridae , Rhabdoviridae , Proteínas de Pez Cebra , Animales , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Infecciones por Rhabdoviridae/veterinaria , Viremia/veterinaria , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , FosfoproteínasRESUMEN
During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.
Asunto(s)
Enfermedades de los Peces , Factores Reguladores del Interferón , Proteínas Quinasas Activadas por Mitógenos , Infecciones por Rhabdoviridae , Ubiquitinación , Proteínas Estructurales Virales , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Rhabdoviridae/genética , Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/virología , Pez Cebra/genética , Pez Cebra/inmunología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Estabilidad Proteica , Proteolisis , Proteínas Estructurales Virales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regulación hacia ArribaRESUMEN
The production of type I interferon is tightly regulated to prevent excessive immune activation. However, the role of selective autophagy receptor SQSTM1 in this regulation in teleost remains unknown. In this study, we cloned the triploid fish SQSTM1 (3nSQSTM1), which comprises 1371 nucleotides, encoding 457 amino acids. qRT-PCR data revealed that the transcript levels of SQSTM1 in triploid fish were increased both in vivo and in vitro following spring viraemia of carp virus (SVCV) infection. Immunofluorescence analysis confirmed that 3nSQSTM1 was mainly distributed in the cytoplasm. Luciferase reporter assay results showed that 3nSQSTM1 significantly blocked the activation of interferon promoters induced by 3nMDA5, 3nMAVS, 3nTBK1, and 3nIRF7. Co-immunoprecipitation assays further confirmed that 3nSQSTM1 could interact with both 3nTBK1 and 3nIRF7. Moreover, upon co-transfection, 3nSQSTM1 significantly inhibited the antiviral activity mediated by TBK1 and IRF7. Mechanistically, 3nSQSTM1 decreased the TBK1 phosphorylation and its interaction with 3nIRF7, thereby suppressing the subsequent antiviral response. Notably, we discovered that 3nSQSTM1 also interacted with SVCV N and P proteins, and these viral proteins may exploit 3nSQSTM1 to further limit the host's antiviral innate immune responses. In conclusion, our study demonstrates that 3nSQSTM1 plays a pivotal role in negatively regulating the interferon signaling pathway by targeting 3nTBK1 and 3nIRF7.
RESUMEN
Glutathione S-transferase P1 (GSTP1), the most ubiquitous member of the GST superfamily, plays vital roles in the detoxification, antioxidant defense, and modulation of inflammatory responses. However, limited studies have been conducted on the function of GSTP1 in antiviral innate immunity. In this study, we have cloned the homolog of GSTP1 in triploid hybrid crucian carp (3nGSTP1) and investigated its regulatory role in the interferon signaling pathway. The open reading frame of 3nGSTP1 is composed of 627 nucleotides, encoding 209 amino acids. In response to spring viremia of carp virus (SVCV) infection, the mRNA level of 3nGSTP1 was up-regulated in the liver, kidney, and caudal fin cell lines (3 nF C) of triploid fish. The knockdown of 3nGSTP1 in 3 nF C improved host cell's antiviral capacity and attenuated SVCV replication. Additionally, overexpression of 3nGSTP1 inhibited the activation of IFN promoters induced by SVCV infection, poly (I:C) stimulation, or the RLR signaling factors. The co-immunoprecipitation assays further revealed that 3nGSTP1 interacts with 3nMAVS. In addition, 3nGSTP1 dose-dependently inhibited 3nMAVS-mediated antiviral activity and reduced 3nMAVS protein level. Mechanistically, 3nGSTP1 promoted ubiquitin-proteasome degradation of MAVS by promoting its K48-linked polyubiquitination. To conclude, our results indicate that GSTP1 acts as a novel inhibitor of MAVS, which negatively regulates the IFN signaling.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Triploidía , Transducción de Señal , Rhabdoviridae/fisiología , Infecciones por Rhabdoviridae/veterinaria , Inmunidad Innata/genética , Poli I-C/farmacología , AntiviralesRESUMEN
The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.
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Carpas , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Rhabdoviridae/fisiología , Reoviridae/fisiología , Infecciones por Rhabdoviridae/veterinaria , Carpas/genética , Carpas/metabolismo , Proteínas de Peces , Inmunidad Innata/genética , Interferones , Mamíferos/metabolismoRESUMEN
Immunoglobulin M (IgM) specifically recognizes various antigens and can activate complement, mediate cytotoxicity, opsonize and agglutinate pathogens to induce phagocytosis, all of which play an important role in immunity. However, the IgM response of common carp (Cyprinus carpio) in the intestinal mucosa after viral infection has not been thoroughly. Therefore, we successfully produced an anti-carp IgM monoclonal antibody and developed a model of viral infection to study the kinetics of immune responses after viral infection. Our results showed that the expression of IL1-ß and Igs were dramatically increased, implying that common carp exhibited a significant innate and adaptive immune response to viral infection. Furthermore, we found that the IgM responses varied between the two infection strategies. At 14 days post-infection (DPI), a significant population of IgM+ B cells were observed in the gut, accompanied by a sharp rise in IgM levels. The immune response to secondary infection started at 7 DPI, suggesting that the IgM response is faster in the gut after re-infection. Importantly, we also explored the variability of different gut compartments to viral infection, and result revealed a stronger immune response in the hindgut than in the foregut and midgut. Overall, our findings indicate that IgM plays an important role in the intestinal immune response following primary and secondary viral infection, in which the hindgut plays a major immune function.
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Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Inmunoglobulina M , Viremia , Inmunidad MucosaRESUMEN
DExD/H-box helicase (DDX) 5 belongs to the DExD/H-box helicase family. DDX family members play differential roles in the regulation of innate antiviral immune response. However, whether DDX5 is involved in antiviral immunity remains unclear. In this study, we found that DDX5 serves as a negative regulator of type I interferon (IFN) response. Overexpression of DDX5 inhibited IFN production induced by Spring viremia of carp virus (SVCV) and poly(I:C) and enhanced virus replication by targeting key elements of the RLR signaling pathway (MAVS, MITA, TBK1, IRF3 and IRF7). Mechanistically, DDX5 directly interacted with TBK1 to promote its autophagy-mediated degradation. Moreover, DDX5 was shown to block the interaction between TRAF3 and TBK1, hence preventing nuclear translocation of IRF3. Together, these data shed light on the roles of DDX5 in regulating IFN response.
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Interferón Tipo I , Proteínas Serina-Treonina Quinasas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Fosforilación , Diclorodifenil Dicloroetileno , Inmunidad Innata , Interferón Tipo I/metabolismo , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , AntiviralesRESUMEN
Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.
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Carpas , Infecciones por Rhabdoviridae/veterinaria , Rhabdoviridae/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Inmunidad Innata , Interferones/metabolismo , Infecciones por Rhabdoviridae/inmunología , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Proteínas de la Matriz Viral/metabolismo , Viremia/veterinariaRESUMEN
IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a-/- knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a-/- and irf2a+/+ cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a-/- cells than irf2a+/+ cells, suggesting that they may contribute to the increased viral resistance of the irf2a-/- cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.
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Enfermedades de los Peces , Factor 2 Regulador del Interferón , Fosfoproteínas , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Enfermedades de los Peces/virología , Interferones/inmunología , Fosfoproteínas/metabolismo , Rhabdoviridae/fisiología , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Viremia , Pez Cebra/virología , Factor 2 Regulador del Interferón/metabolismo , Técnicas de Inactivación de Genes , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT1 , Replicación ViralRESUMEN
Spring viremia of carp virus (SVCV) is a lethal freshwater pathogen of cyprinid fish that has caused significant economic losses to aquaculture. To reduce the economic losses caused by SVCV, its pathogenic mechanism needs to be studied more thoroughly. Here, we report for the first time that SVCV infection of Epithelioma papulosum cyprini (EPC) cells can induce cellular autophagy and apoptosis through endoplasmic reticulum stress. The presence of autophagic vesicles in infected EPC cells was shown by transmission electron microscopy. Quantitative fluorescence PCR and Western blot results showed that p62 mRNA expression was decreased, and the expression of Beclin1 and LC3 mRNA was increased. The p62 protein was decreased, and the Beclin1 protein and LC3 were increased in the endoplasmic reticulum stress activation state. To further clarify the mode of death of SVCV-infected EPC cells, we examined caspase3, caspase9, BCL-2, and Bax mRNA, which showed that they were all increased. Apoptosis of SVCV-infected cells increased upon activation of endoplasmic reticulum stress. Our results suggest that endoplasmic reticulum stress can regulate SVCV infection-induced autophagy and apoptosis. The results of this study provide theoretical data for the pathogenesis of SVCV and lay the foundation for future drug development and vaccine construction.
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Carcinoma , Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Animales , Viremia , Beclina-1 , Apoptosis , AutofagiaRESUMEN
Herbal immunomodulators are an important part of prevention and control on viral diseases in aquaculture because of their propensity to improve immunity in fish. The present study was conducted to evaluate the immunomodulatory effect and antiviral activity of a synthesized derivative (serial number: LML1022) against spring viremia of carp virus (SVCV) infection in vitro and in vivo. The antiviral data suggested that LML1022 at 100 µM significantly inhibited the virus replication in epithelioma papulosum cyprini (EPC) cells, and may completely inhibit the infectivity of SVCV virion particles to fish cells by affecting the viral internalization. The results in the related stability of water environments also demonstrated that LML1022 had an inhibitory half-life of 2.3 d at 15 °C, which would facilitate rapid degradation of LML1022 in aquaculture application. For in vivo study, the survival rate of SVCV-infected common carp was increased 30% at least under continuous oral injection of LML1022 at 2.0 mg/kg for 7 d treatment. Additionally, pretreatment of LML1022 on fish prior to SVCV infection also obviously reduced the viral loads in vivo as well as an improved survival rate, showing that LML1022 was potential as an immunomodulator. As an immune response, LML1022 significantly upregulated the immune-related gene expression including IFN-γ2b, IFN-I, ISG15 and Mx1, indicating that its dietary administration may improve the resistance of common carp against SVCV infection.
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Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/tratamiento farmacológico , Rhabdoviridae/fisiología , Antivirales/farmacología , Antivirales/uso terapéutico , Factores Inmunológicos/farmacología , Adyuvantes Inmunológicos/farmacología , Viremia/tratamiento farmacológicoRESUMEN
Interleukin (IL) 27 is a member of the IL-12 family and is a heterodimeric cytokine composed of IL-27A and Epstein-Barr virus-induced 3 (EBI3). It plays an important role in regulating inflammation and cancer progression. IL-27A not only functions by dimerizing with EBI3 but also acts alone. Here, we report that IL-27A and EBI3 suppress spring viremia of carp virus (SVCV) replication in zebrafish. Expression analysis reveals that il-27a and ebi3 were significantly upregulated in the ZF4 cells by SVCV and poly(I:C), and in the zebrafish caudal fin (ZFIN) cells overexpressed with SVCV genes. Interestingly, il-27a and ebi3 were not modulated by IFNφ1, indicating that they are not IFN stimulated genes (ISGs). Furthermore, overexpression of IL-27A and EBI3 alone inhibited SVCV replication in the EPC cells, but less potent than co-expression of IL-27A and EBI3. Intriguingly, IL-27A could not induce the expression of irf3, ifn, isg15 and mx1. Taken together, our results demonstrate that IL-27A and EBI3 activate innate antiviral response in an IFN independent manner in zebrafish.
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Enfermedades de los Peces , Interleucina-27 , Infecciones por Rhabdoviridae , Rhabdoviridae , Pez Cebra , Animales , Infecciones por Virus de Epstein-Barr , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Herpesvirus Humano 4/metabolismo , Interleucina-27/genética , Interleucinas/genética , Rhabdoviridae/fisiología , Infecciones por Rhabdoviridae/veterinaria , Viremia , Replicación Viral , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Lysine methylation is a post-translational modification of histone and non-histone proteins and affects numerous cellular processes. The actin histidine methyltransferase SET domain containing 3 (SETD3) is a member of the protein lysine methyltransferase (PKMT) family which catalyse the addition of methyl groups to lysine residues. However, the role of SETD3 in virus-mediated innate immune responses has rarely been investigated. In this study, zebrafish SETD3 was shown to be induced by poly(I:C) and spring viremia of carp virus (SVCV) and inhibited virus infection. Further, it was found that SETD3 directly interacted with SVCV phosphoprotein (SVCV P) in the cytoplasm of EPC cells, initiating ubiquitination to degrade the SVCV P protein via proteasomal pathway. Interestingly, mutants lacking the SET and RSB domains were able to promote degradation of SVCV P, indicating that they are not required for SETD3 mediated degradation of SVCV P. Taken together, our study demonstrates that SETD3 is an antiviral factor which limits virus replication by promoting ubiquitination of viral phosphoprotein and subsequent protein degradation.
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Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Viremia , Fosfoproteínas/genética , Carpas/genética , Carpas/metabolismo , Lisina , Rhabdoviridae/fisiología , UbiquitinaciónRESUMEN
Rhomboid domain-containing protein 3 (Rhbdd3) is a member of the rhomboid family, which can modulate the innate immune response in mammals. Nonetheless, the function and regulatory mechanism of fish Rhbdd3 during viral infection have not been characterized. In this study, Rhbdd3 was firstly cloned from common carp (Cyprinus carpio) and nominated as CcRhbdd3. Phylogenetically characterization showed that CcRhbdd3 shared a relatively long evolutionary distance with its mammalian homologs. In vivo experiment demonstrated that spring viraemia of carp virus (SVCV) infection promoted the expression of CcRhbdd3 in the liver, spleen, kidney and muscle tissues. Furthermore, overexpression of CcRhbdd3 significantly inhibited SVCV propagation, whereas knockdown of CcRhbdd3 markedly promoted SVCV replication in susceptible cells. RNA-seq and following validation showed that CcRhbdd3 overexpression upregulated the expression of several RIG-I signaling related genes, including TRIM25, TRAF2, MDA5, LGP2, IFN1, IFN3, RIG-I, IRF3 and ISG15. Moreover, CcRhbdd3 promoted the expression of NF-κB, a central immune regulator. Subcellular localization experiments showed that CcRhbdd3 was primarily distributed in the cytoplasm and co-localized with Rab5 in the early endosomes. Truncation experiments further demonstrated that the C-terminus containing the ubiquitin-binding associated domain, was crucial for both the subcellular localization and antiviral activity of CcRhbdd3. The findings in this study provide new insight into the host antiviral mechanism against aquatic RNA virus infection, and will facilitate the development of therapeutic strategies for the infection of SVCV.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Carpas/metabolismo , Proteínas de Peces/química , Rhabdoviridae/fisiología , Inmunidad Innata/genética , Transducción de Señal , Antivirales , Mamíferos/metabolismoRESUMEN
Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election system to minimise stress and the vaccination costs associated to injection. However, administration of prophylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminary i.p injection challenge. The G3 domain of SVCV glycoprotein G was overexpressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or ifngr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and ifngr1 (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality.
Asunto(s)
Carpas , Enfermedades de los Peces , Nanopartículas , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Pez Cebra , Viremia , Antivirales/uso terapéutico , Escherichia coli , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/tratamiento farmacológico , Vacunas de SubunidadRESUMEN
Spring viraemia of carp virus (SVCV) is a fierce pathogen causing high mortality in the common carp. At present, the treatment of spring viraemia of carp (SVC) is limited. Innate immunity is the host's first line of defense against microbial pathogens. Retinoic acid-inducible gene I (RIG-I) activation plays an essential role in the antiviral immune response. Virus infection can activate the RIG-I signaling and induce the production of interferon (IFN) and the expression of IFN-stimulated genes (ISGs). STUB1 (STIP1 homology and U-box containing protein 1) is a highly conserved cytoplasmic protein. This protein is known to exist widely in many biological systems and plays an important role in the process of immune regulation, but little is known in fish. To explore the immune function of STUB1 in fish, STUB1 gene was cloned from zebrafish and analyzed in this study. Zebrafish STUB1 showed 77% and 79% amino acid sequence homology with those from human and mouse, respectively. The amino acid sequence of zebrafish STUB1 contains three TPR domains and one U-box domain. Subcellular localization study revealed that STUB1 is located in the cytoplasm. And overexpression of zebrafish STUB1 resulted in the activation of the transcription of IFN1 and ISGs. Functional analysis showed that STUB1 was able to activate RIG-I signaling, and promote the expression of RIG-I, but STUB1 can degrade RIG-I in mammals. The proliferation of SVCV was significantly inhibited after the overexpression of STUB1 and N-terminal TPR domain of STUB1 in EPC cells. And through secondary structure analysis, overexpression of the mutant of STUB1 110 amino acid resulted in weakened antiviral ability. The expression of STUB1 was attenuated by poly(I:C) treatment and SVCV infection. In summary, this study demonstrated for the first time that STUB1 can induce the production of IFN, enhance the expression of ISGs by promoting the expression of RIG-I and inhibiting viral replication in fish. These findings may form the essential basis for the development of antiviral targets and drugs.
Asunto(s)
Carpas , Rhabdoviridae , Animales , Antivirales/metabolismo , Antivirales/farmacología , Carpas/metabolismo , Inmunidad Innata/genética , Mamíferos/metabolismo , Ratones , Rhabdoviridae/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Common carp (Cyprinus carpio L.) is one of the most widely cultivated fish in China. Spring viraemia of carp virus (SVCV) is a highly pathogenic virus and has often caused excessive losses in carp pond fisheries. Innate immune play important roles against virus infection. To better understand the immune response of common carp against SVCV infection, transcriptome analysis was performed using the Illumina Novaseq 6000 platform. It was showed that a total of 3953 differentially expressed unigenes were identified, and the RLR signaling pathway were significantly enriched after SVCV infection. Subsequently, the role of RLR signaling pathway in SVCV infection was studied. The results showed that common carp RIG-I (CcRIG-I) and TRIM25 (CcTRIM25) significantly decreased the replication of SVCV by inducing the phosphorylation of TBK1, IRF3 and p65 and the expression of ifn-1, viperin, isg15 and mx. Further studies illustrated that CcTRIM25 could positive regulate CcRIG-I mediated downstream signaling pathway. Finally, the mechanism of CcTRIM25 promoting CcRIG-I-mediated signaling was investigated. CcTRIM25 could interact with the caspase activation and recruitment domain (CARD) of CcRIG-I and promoted K63-linked polyubiquitination of CcRIG-I. Altogether, the study revealed a mechanism of CcTRIM25 regulating CcRIG-I mediated immune response in SVCV infection.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Carpas/genética , Rhabdoviridae/fisiología , Transducción de Señal , ViremiaRESUMEN
Spring viremia of carp virus (SVCV), is a lethal freshwater pathogen of cyprinid fish, and Cyprinus carpio koi is a primary host species. The virus was initially described in the 1960s after outbreaks occurred in Europe, but a global expansion of SVCV has been ongoing since the late 1990s. Genetic typing of SVCV isolates separates them into 4 genotypes that are correlated with geographic origin: Ia (Asia), Ib and Ic (Eastern Europe), and Id (Central Europe). We compared infectivity and virulence of 8 SVCV strains, including 4 uncharacterized Chinese Ia isolates and representatives of genotypes Ia-d in 2 morphologically distinct varieties of koi: long-fin semi-scaled Beni Kikokuryu koi and short-fin fully scaled Sanke koi. Mortality ranged from 4 to 82% in the Beni Kikokuryu koi and 0 to 94% in the Sanke koi following immersion challenge. Genotype Ia isolates of Asian origin had a wide range in virulence (0-94%). Single isolates representing the European genotypes Ib and Ic were moderately virulent (38-56%). Each virus strain produced similar levels of mortality in both koi breeds, with the exception of the SVCV Id strain that appeared to have both moderate and high virulence phenotypes (60% in Beni Kikokuryu koi vs. 87% in Sanke koi). Overall SVCV strain virulence appeared to be a dominant factor in determining disease outcomes, whereas intraspecies variation, based on koi variety, had less of an impact. This study is the first side-by-side comparison of Chinese SVCV isolates and genotype Ia-d strain virulence in a highly susceptible host.