RESUMEN
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
Gene regulatory networks (GRNs) are crucial for understanding organismal molecular mechanisms and processes. Construction of GRN in the epithelioma papulosum cyprini (EPC) cells of cyprinid fish by spring viremia of carp virus (SVCV) infection helps understand the immune regulatory mechanisms that enhance the survival capabilities of cyprinid fish. Although many computational methods have been used to infer GRNs, specialized approaches for predicting the GRN of EPC cells following SVCV infection are lacking. In addition, most existing methods focus primarily on gene expression features, neglecting the valuable network structural information in known GRNs. In this study, we propose a novel supervised deep neural network, named MEFFGRN (Matrix Enhancement- and Feature Fusion-based method for Gene Regulatory Network inference), to accurately predict the GRN of EPC cells following SVCV infection. MEFFGRN considers both gene expression data and network structure information of known GRN and introduces a matrix enhancement method to address the sparsity issue of known GRN, extracting richer network structure information. To optimize the benefits of CNN (Convolutional Neural Network) in image processing, gene expression and enhanced GRN data were transformed into histogram images for each gene pair respectively. Subsequently, these histograms were separately fed into CNNs for training to obtain the corresponding gene expression and network structural features. Furthermore, a feature fusion mechanism was introduced to comprehensively integrate the gene expression and network structural features. This integration considers the specificity of each feature and their interactive information, resulting in a more comprehensive and precise feature representation during the fusion process. Experimental results from both real-world and benchmark datasets demonstrate that MEFFGRN achieves competitive performance compared with state-of-the-art computational methods. Furthermore, study findings from SVCV-infected EPC cells suggest that MEFFGRN can predict novel gene regulatory relationships.
Asunto(s)
Enfermedades de los Peces , Redes Reguladoras de Genes , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Rhabdoviridae/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/virología , Infecciones por Rhabdoviridae/genética , Infecciones por Rhabdoviridae/virología , Carpas/genética , Carpas/virología , Biología Computacional/métodos , Redes Neurales de la Computación , Cyprinidae/genéticaRESUMEN
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
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.
Asunto(s)
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
[This corrects the article DOI: 10.3389/fimmu.2022.1031962.].
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
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathways are required to be tightly controlled to initiate host innate immune responses. Fish mitochondrial antiviral signaling (mavs) is a key determinant in the RLR pathway, and its ubiquitination is associated with mavs activation. Here, we identified the zebrafish E3 ubiquitin ligase Speckle-type BTB-POZ protein (spop) negatively regulates mavs-mediated the type I interferon (IFN) responses. Consistently, overexpression of zebrafish spop repressed the activity of IFN promoter and reduced host ifn transcription, whereas knockdown spop by small interfering RNA (siRNA) transfection had the opposite effects. Accordingly, overexpression of spop dampened the cellular antiviral responses triggered by spring viremia of carp virus (SVCV). A functional domain assay revealed that the N-terminal substrate-binding MATH domain regions of spop were necessary for IFN suppression. Further assays indicated that spop interacts with mavs through the C-terminal transmembrane (TM) domain of mavs. Moreover, zebrafish spop selectively promotes K48-linked polyubiquitination and degradation of mavs through the lysosomal pathway to suppress IFN expression. Our findings unearth a post-translational mechanism by which mavs is regulated and reveal a role for spop in inhibiting antiviral innate responses.
Asunto(s)
Transducción de Señal , Pez Cebra , Animales , Ubiquitinación , Inmunidad Innata , AntiviralesRESUMEN
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
Interleukin-10 (IL-10) is a pleiotropic cytokine with both immune enhancement and immunosuppression activities, but the main role is immunosuppression and anti-inflammatory ability. In order to use the immunosuppressive function of IL-10, many viruses, such as SARS-CoV-2, hepatitis B virus and EB virus, can evade the host's immune surveillance and clearance by increasing the expression of host IL-10. However, it has not been reported whether the aquatic animal infection virus can upregulate the expression of host IL-10 and the mechanisms are still unknown. Spring viremia of carp (SVC) is a fatal viral disease for many fish species and is caused by spring viremia of carp virus (SVCV). This disease has caused significant economic losses in the aquaculture industry worldwide. In this study, the expression of carp IL-10 with or without infection of SVCV in epithelioma papulosum cyprinid (EPC) cells, carp head kidney (cHK) primary cells and common carp tissues were analyzed using RT-PCR and ELISA. The results show that SVCV infection induced carp IL-10 mRNA and protein expression, both in vitro and in vivo. However, the upregulation of carp IL-10 by SVCV was hindered by specific inhibitors of the JAK inhibitor (CP-690550), STAT3 inhibitor (STA-21), NF-κB inhibitor (BAY11-7082) and p38 MAPK (mitogen-activated protein kinase) inhibitor (SB202190), but not JNK inhibitor (SP600125). Furthermore, the results demonstrated that JAK1, JAK2, JAK3, TYK2 and STAT5 played important roles in carp IL-10 production induced by SVCV infection. Taken together, SVCV infection significantly induced carp IL-10 expression and the upregulation trigged in JAK-STAT, NF-κB and p38MAPK pathways. To our knowledge, this is the first time that a fish infection virus upregulated the host IL-10 expression through the JAK-STAT, NF-κB and p38MAPK pathways. Altogether, fish viruses may have a similar mechanism as human or other mammalian viruses to escape host immune surveillance and clearance.
RESUMEN
Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.
Asunto(s)
Carpas , MicroARNs , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/tratamiento farmacológico , Pez Cebra/genética , Viremia , MicroARNs/genética , Rhabdoviridae/genéticaRESUMEN
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
Mammalian heterogeneous nuclear ribonucleoproteins M (hnRNPM) is a critical splicing regulatory protein that has been reported to negatively regulate the RLR signaling pathway by impairing the binding of RIG-I and MDA5 to viral RNA. To explore the role of hnRNPM in the antiviral innate immune response in teleost fish, the hnRNPM homologue of triploid fish (3nhnRNPM) has been cloned and identified in this paper. The CDS of 3nhnRNPM gene is composed of 2016 nucleotides and encodes 671 amino acids. 3nhnRNPM migrated around 71 kDa in immunoblotting assay and was mainly detected in the nucleus in nucleo-cytoplasmic separation assay and immunofluorescent staining test. When 3nhnRNPM and 3nIRF7 were co-expressed in EPC cells, 3nhnRNPM significantly reduced the 3nIRF7-induced interferon (IFN) promoter transcription. Correspondingly, the mRNA levels of the SVCV-M, -N, -P, and -G genes were noteworthily enhanced, but the transcription levels of epcIFNφ1, epcMx1, epcPKR, and epcISG15 were dramatically decreased. Additionally, the knockdown of 3nhnRNPM resulted in restricted SVCV replication and enhanced host cell antiviral activity. Furthermore, the association between 3nhnRNPM and 3nIRF7 has been identified by the co-immunoprecipitation assay. In addition, we found that 3nIRF7 was detained in the nucleus when co-expressed with 3nhnRNPM. To sum up, our data supported the conclusion that 3nhnRNPM suppressed 3nIRF7-mediated IFN signaling in the antiviral innate immunity.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Rhabdoviridae/fisiología , Reoviridae/fisiología , Antivirales , Triploidía , Carpas/genética , Carpas/metabolismo , Proteínas de Peces/metabolismo , Inmunidad Innata/genética , Transducción de Señal , MamíferosRESUMEN
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.
Asunto(s)
Carcinoma , Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Animales , Viremia , Beclina-1 , Apoptosis , AutofagiaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
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 core binding factor subunit beta (CBFß) is a transcription factor that forms a complex with virial proteins to promote viral infection. In this study, we identified a CBFß homolog from zebrafish (zfCBFß) and characterized the biological activity. The deduced zfCBFß protein was highly similar to orthologs from other species. The zfcbfß gene was constitutively expressed in tissues and was induced in immune tissues after infection with spring viremia carp virus (SVCV) and stimulation with poly(I:C). Interestingly, zfcbfß is not induced by type I interferons. Overexpression of zfcbfß induced tnfα expression but inhibited isg15 expression. Also, overexpression of zfcbfß significantly increased SVCV titer in the EPC cells. Co-immunoprecipitation assay revealed that zfCBFß interacts with SVCV phosphoprotein (SVCVP) and host p53, resulting in the increased stability of zfCBFß. Our results provide evidence that CBFß is targeted by virus to suppress host antiviral response.
Asunto(s)
Carpas , Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Rhabdoviridae/fisiología , Pez Cebra , Viremia , Replicación ViralRESUMEN
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.
Asunto(s)
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
In mammals, interferon (IFN)-stimulated genes (ISGs) play important roles in restricting the replication of viruses. However, the functions of many ISGs have not been investigated in fish. In this study, eight isg12 homologs (termed isg12.1-8) were identified in zebrafish and all contain a typical ISG12 family domain rich of hydrophobic amino acid residues. Isg12.1-7 were significantly induced in the ZF4 cells by poly(I:C) and IFNφ1, and in the kidney and spleen after infection with spring viremia of carp virus (SVCV). In the EPC cells, overexpression of isg12.1 inhibited SVCV replication. Further, it was found that zebrafish ISG12.1 interacted with SVCV phosphoprotein (SVCV-P) and promoted SVCV-P degradation which could be attenuated by 3-MA and CQ (autophagy inhibitors). Our results indicate that zebrafish ISG12.1 restricts viral replication by targeting viral phosphoprotein for degradation.
Asunto(s)
Enfermedades de los Peces , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Pez Cebra , Fosfoproteínas/genética , Replicación Viral , MamíferosRESUMEN
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.