RESUMO
Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.
Assuntos
Proteínas do Capsídeo , Doenças dos Peixes , Evasão da Resposta Imune , Imunidade Inata , Nodaviridae , Nucleotidiltransferases , Infecções por Vírus de RNA , Transdução de Sinais , Replicação Viral , Animais , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/metabolismo , Interferons/metabolismo , Interferons/imunologia , Bass/imunologia , Bass/virologia , Bass/metabolismo , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologiaRESUMO
Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques.
Assuntos
Bass , Doenças dos Peixes , Proteínas de Peixes , Imunidade Inata , Nodaviridae , Infecções por Vírus de RNA , Replicação Viral , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Imunidade Inata/genética , Nodaviridae/fisiologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Bass/imunologia , Filogenia , Regulação da Expressão Gênica/imunologia , Sequência de Aminoácidos , Alinhamento de Sequência/veterinária , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/imunologia , Perfilação da Expressão Gênica/veterináriaRESUMO
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
Assuntos
Infecções por Vírus de RNA , Grânulos de Estresse , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Grânulos Citoplasmáticos/metabolismo , Proteína DEAD-box 58/metabolismo , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Imunidade Inata , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores Imunológicos/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Grânulos de Estresse/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.
Assuntos
Encéfalo , Doenças dos Peixes , Macrófagos , Nodaviridae , Infecções por Vírus de RNA , Animais , Macrófagos/imunologia , Macrófagos/virologia , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Encéfalo/virologia , Encéfalo/imunologia , Encéfalo/patologia , Nodaviridae/fisiologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Quimiocina CXCL11 , Receptores CXCR3/metabolismo , Bass/imunologia , Bass/virologia , Transdução de Sinais , Citocinas/metabolismo , Citocinas/imunologia , Proteínas de Peixes/imunologia , Proteínas de Peixes/genéticaRESUMO
While macrophage is one of the major type I interferon (IFN-I) producers in multiple tissues during viral infections, it also serves as an important target cell for many RNA viruses. However, the regulatory mechanism for the IFN-I response of macrophages to respond to a viral challenge is not fully understood. Here we report ADAP, an immune adaptor protein, is indispensable for the induction of the IFN-I response of macrophages to RNA virus infections via an inhibition of the conjugation of ubiquitin-like ISG15 (ISGylation) to RIG-I. Loss of ADAP increases RNA virus replication in macrophages, accompanied with a decrease in LPS-induced IFN-ß and ISG15 mRNA expression and an impairment in the RNA virus-induced phosphorylation of IRF3 and TBK1. Moreover, using Adap-/- mice, we show ADAP deficiency strongly increases the susceptibility of macrophages to RNA-virus infection in vivo. Mechanically, ADAP selectively interacts and functionally cooperates with RIG-I but not MDA5 in the activation of IFN-ß transcription. Loss of ADAP results in an enhancement of ISGylation of RIG-I, whereas overexpression of ADAP exhibits the opposite effect in vitro, indicating ADAP is detrimental to the RNA virus-induced ISGylation of RIG-I. Together, our data demonstrate a novel antagonistic activity of ADAP in the cell-intrinsic control of RIG-I ISGylation, which is indispensable for initiating and sustaining the IFN-I response of macrophages to RNA virus infections and replication.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteína DEAD-box 58 , Interferon Tipo I , Macrófagos , Camundongos Knockout , Infecções por Vírus de RNA , Ubiquitinas , Animais , Macrófagos/virologia , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/genética , Proteína DEAD-box 58/metabolismo , Interferon Tipo I/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Citocinas/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Receptores Imunológicos/metabolismo , Interferon beta/metabolismo , Vírus de RNA/imunologia , Fator Regulador 3 de Interferon/metabolismoRESUMO
Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.
Assuntos
Doenças dos Peixes , Infecções por Rhabdoviridae , Rhabdoviridae , Animais , Doenças dos Peixes/virologia , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/prevenção & controle , Rhabdoviridae/fisiologia , Antivirais/farmacologia , Antivirais/química , Percas , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Peptídeos/farmacologia , Peptídeos/química , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/prevenção & controleRESUMO
The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.
Assuntos
Doenças dos Peixes , Linguados , Nodaviridae , Infecções por Vírus de RNA , Vacinas de Produtos Inativados , Vacinas Virais , Animais , Doenças dos Peixes/prevenção & controle , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Linguados/imunologia , Linguados/virologia , Nodaviridae/imunologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/prevenção & controle , Infecções por Vírus de RNA/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinação/veterinária , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes de Vacinas/administração & dosagemRESUMO
Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.
Assuntos
Antígenos CD40 , Infecções por Vírus de RNA , Vírus de RNA , Animais , Camundongos , Antígenos CD40/metabolismo , Interferon gama , Macrófagos , Infecções por Vírus de RNA/imunologiaRESUMO
Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.
Assuntos
Bass , Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Bass/virologia , Proteínas do Capsídeo/metabolismo , Epitopos de Linfócito B/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Epitopos Imunodominantes , Necrose , Nodaviridae/fisiologia , Infecções por Vírus de RNA/imunologiaRESUMO
XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Fator Regulador 1 de Interferon , Infecções por Vírus de RNA , Vírus de RNA , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Humanos , Imunidade Inata , Fator Regulador 1 de Interferon/imunologia , Camundongos , Camundongos Knockout , Infecções por Vírus de RNA/imunologia , Replicação ViralRESUMO
Bats are important hosts for various zoonotic viral diseases. However, they rarely show signs of disease infection with such viruses. As the first line for virus control, the innate immune system of bats attracted our full attention. In this study, the Tadarida brasiliensis MDA5 gene (batMDA5), a major sensor for anti-RNA viral infection, was first cloned, and its biological functions in antiviral innate immunity were identified. Bioinformatics analysis shows that the amino acid sequence of batMDA5 is poorly conserved among species, and it is evolutionarily closer to humans. The mRNA of batMDA5 was significantly upregulated in Newcastle disease virus (NDV), avian influenza virus (AIV), and vesicular stomatitis virus (VSV)-infected bat TB 1 Lu cells. Overexpression of batMDA5 could activate IFNß and inhibit vesicular stomatitis virus (VSV-GFP) replication in TB 1 Lu cells, while knockdown of batMDA5 yielded the opposite result. In addition, we found that the CARD domain was essential for MDA5 to activate IFNß by constructing MDA5 domain mutant plasmids. These results indicated that bat employs a conserved MDA5 gene to trigger anti-RNA virus innate immune response. This study helps understand the biological role of MDA5 in innate immunity during evolution.
Assuntos
Quirópteros , Imunidade Inata , Helicase IFIH1 Induzida por Interferon , Infecções por Vírus de RNA , Animais , Quirópteros/imunologia , Vírus da Influenza A , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta , Infecções por Vírus de RNA/imunologia , Vírus de RNARESUMO
Retinoic acid-inducible-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) genes encode essential cytosolic receptors mediating antiviral immunity against viruses. Here, we show that OTUD3 has opposing role in response to RNA and DNA virus infection by removing distinct types of RIG-I/MDA5 and cGAS polyubiquitination. OTUD3 binds to RIG-I and MDA5 and removes K63-linked ubiquitination. This serves to reduce the binding of RIG-I and MDA5 to viral RNA and the downstream adaptor MAVS, leading to the suppression of the RNA virus-triggered innate antiviral responses. Meanwhile, OTUD3 associates with cGAS and targets at Lys279 to deubiquitinate K48-linked ubiquitination, resulting in the enhancement of cGAS protein stability and DNA-binding ability. As a result, Otud3-deficient mice and zebrafish are more resistant to RNA virus infection but are more susceptible to DNA virus infection. These findings demonstrate that OTUD3 limits RNA virus-triggered innate immunity but promotes DNA virus-triggered innate immunity.
Assuntos
Infecções por Vírus de DNA , Imunidade Inata , Infecções por Vírus de RNA , Proteases Específicas de Ubiquitina , Animais , Proteína DEAD-box 58/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA , Enzimas Desubiquitinantes , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Nucleotidiltransferases , Infecções por Vírus de RNA/imunologia , Vírus de RNA , RNA Viral/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Peixe-Zebra/metabolismoRESUMO
The RNA-binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFNs). This signal transduction occurs at endoplasmic reticulum (ER)mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, as one of the proteins recruited to membranes at ERmitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We found that following RNA virus infection, UFL1 is recruited to the membrane-targeting protein 143-3ε and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 143-3ε has an increase in UFM1 conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 143-3ε with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus the downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a posttranslational control for IFN induction.
Assuntos
Proteína DEAD-box 58 , Interferons , Infecções por Vírus de RNA , RNA Viral , Receptores Imunológicos , Ubiquitina-Proteína Ligases , Proteínas 14-3-3/metabolismo , Proteína DEAD-box 58/metabolismo , Humanos , Imunidade Inata , Interferons/metabolismo , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/imunologia , RNA Viral/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Respiratory viruses present major public health challenges, as evidenced by the 1918 Spanish Flu, the 1957 H2N2, 1968 H3N2, and 2009 H1N1 influenza pandemics, and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestations at the cell and tissue levels is vital to understanding the mechanisms of immunopathology and to developing strain-independent treatments. Here, we present a novel spatialized multicellular computational model of RNA virus infection and the type-I interferon-mediated antiviral response that it induces within lung epithelial cells. The model is built using the CompuCell3D multicellular simulation environment and is parameterized using data from influenza virus-infected cell cultures. Consistent with experimental observations, it exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. The model suggests that modifying the activity of signaling molecules in the JAK/STAT pathway or altering the ratio of the diffusion lengths of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of plaque growth arrest on diffusion lengths highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro measurement of these diffusion coefficients. Sensitivity analyses under conditions leading to continuous or arrested plaque growth found that plaque growth is more sensitive to variations of most parameters and more likely to have identifiable model parameters when conditions lead to plaque arrest. This result suggests that cytokine assay measurements may be most informative under conditions leading to arrested plaque growth. The model is easy to extend to include SARS-CoV-2-specific mechanisms or to use as a component in models linking epithelial cell signaling to systemic immune models.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Interferons , Infecções por Vírus de RNA , Vírus de RNA , Replicação Viral , Células Cultivadas , Biologia Computacional , Células Epiteliais/imunologia , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Interferons/metabolismo , Pulmão/citologia , Pulmão/imunologia , Modelos Biológicos , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/imunologia , Vírus de RNA/fisiologia , Replicação Viral/imunologia , Replicação Viral/fisiologiaRESUMO
In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.
Assuntos
Doenças dos Peixes , Microglia/imunologia , Doenças Neuroinflamatórias , Infecções por Vírus de RNA , Animais , Comportamento Animal , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/virologia , Ingestão de Alimentos , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Expressão Gênica , Comportamento de Doença , Locomoção , Macrófagos/imunologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/veterinária , Doenças Neuroinflamatórias/virologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/patologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , Carga Viral , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Tumor necrosis factor receptor-associated factors (TRAFs) play important roles in the biological processes of immune regulation, the inflammatory response, and apoptosis. TRAF4 belongs to the TRAF family and plays a major role in many biological processes. Compared with other TRAF proteins, the functions of TRAF4 in teleosts have been largely unknown. In the present study, the TRAF4 homologue (EcTRAF4) of the orange-spotted grouper was characterized. EcTRAF4 consisted of 1413 bp encoding a 471-amino-acid protein, and the predicted molecular mass was 54.27 kDa. EcTRAF4 shares 99.79% of its identity with TRAF4 of the giant grouper (E. lanceolatus). EcTRAF4 transcripts were ubiquitously and differentially expressed in all the examined tissues. EcTRAF4 expression in GS cells was significantly upregulated after stimulation with red-spotted grouper nervous necrosis virus (RGNNV). EcTRAF4 protein was distributed in the cytoplasm of GS cells. Overexpressed EcTRAF4 promoted RGNNV replication during viral infection in vitro. Yeast two-hybrid and coimmunoprecipitation assays showed that EcTRAF4 interacted with the coat protein (CP) of RGNNV. EcTRAF4 inhibited the activation of IFN3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB). Overexpressed EcTRAF4 also reduced the expression of interferon (IFN)-related molecules and pro-inflammatory factors. Together, these results demonstrate that EcTRAF4 plays crucial roles in RGNNV infection.
Assuntos
Bass/virologia , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Nodaviridae/fisiologia , Infecções por Vírus de RNA/veterinária , Fator 4 Associado a Receptor de TNF/metabolismo , Replicação Viral , Animais , Bass/genética , Bass/imunologia , Bass/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/metabolismo , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Fator 4 Associado a Receptor de TNF/genéticaRESUMO
The antiviral innate immune responses are crucial steps during host defense and must be strictly regulated, but the molecular mechanisms of control remain unclear. In this study, we report increased expression of human ATPase Na+/K+ transporting subunit ß 1(ATP1B1) after DNA and RNA virus infections. We found that the expression of ATP1B1 can inhibit viral replication and increase the levels of IFNs, IFN-stimulated genes, and inflammatory cytokines. Knockdown of ATP1B1 by specific short hairpin RNA had the opposite effects. Upon viral infection, ATP1B1 was induced, interacted with TRAF3 and TRAF6, and potentiated the ubiquitination of these proteins, leading to increased phosphorylation of downstream molecules, including TGF-ß-activated kinase 1 (TAK1) and TANK-binding kinase 1 (TBK1). These results reveal a previously unrecognized role of ATP1B1 in antiviral innate immunity and suggest a novel mechanism for the induction of IFNs and proinflammatory cytokines during viral infection.
Assuntos
Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , ATPase Trocadora de Sódio-Potássio/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Regulação para Cima/imunologia , Animais , Células Cultivadas , Chlorocebus aethiops , Infecções por Vírus de DNA/imunologia , Vírus de DNA/imunologia , Humanos , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , ATPase Trocadora de Sódio-Potássio/genética , Ubiquitinação/imunologia , Replicação ViralRESUMO
A systematic and flexible immunoregulatory network is required to ensure the proper outcome of antiviral immune signaling and maintain homeostasis during viral infection. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a novel immunoregulatory protein, has been extensively studied in inflammatory response, apoptosis, and cancer. However, the function of TIPE2 in antiviral innate immunity is poorly clarified. In this study, we reported that the expression of TIPE2 declined at the early period and then climbed up in macrophages under RNA virus stimulation. Knockout of TIPE2 in the macrophages enhanced the antiviral capacity and facilitated type I interferon (IFN) signaling after RNA viral infection both in vitro and in vivo. Consistently, overexpression of TIPE2 inhibited the production of type I IFNs and pro-inflammatory cytokines, and thus promoted the viral infection. Moreover, TIPE2 restrained the activation of TBK1 and IRF3 in the retinoic acid inducible gene-I (RIG-I)-like receptors (RLR) signaling pathway by directly interacting with retinoic acid inducible gene-I (RIG-I). Taken together, our results suggested that TIPE2 suppresses the type I IFN response induced by RNA virus by targeting RIG-I and blocking the activation of downstream signaling. These findings will provide new insights to reveal the immunological function of TIPE2 and may help to develop new strategies for the clinical treatment of RNA viral infections.
Assuntos
Proteína DEAD-box 58/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macrófagos/imunologia , Infecções por Vírus de RNA/imunologia , Receptores Imunológicos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Humanos , Imunidade Inata , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Replicação ViralRESUMO
Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper (Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens, respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1-45), IMD (aa 46-67), CIL (aa 68-93), TMD (aa 94-119), and CTD (aa 120-131). In vitro, the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections.
Assuntos
Antígenos de Diferenciação/metabolismo , Infecções por Vírus de DNA/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Iridoviridae/fisiologia , Nodaviridae/fisiologia , Perciformes/imunologia , Infecções por Vírus de RNA/imunologia , Animais , Antígenos de Diferenciação/genética , Células Cultivadas , Clonagem Molecular , Proteínas de Peixes/genética , Metabolismo dos Lipídeos , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Regulação para Cima , Internalização do Vírus , Replicação ViralRESUMO
N6-Methyladenosine (m6A) is the most abundant internal RNA modification catalyzed by host RNA methyltransferases. As obligate intracellular parasites, many viruses acquire m6A methylation in their RNAs. However, the biological functions of viral m6A methylation are poorly understood. Here, we found that viral m6A methylation serves as a molecular marker for host innate immunity to discriminate self from nonself RNA and that this novel biological function of viral m6A methylation is universally conserved in several families in nonsegmented negative-sense (NNS) RNA viruses. Using m6A methyltransferase (METTL3) knockout cells, we produced m6A-deficient virion RNAs from the representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae and found that these m6A-deficient viral RNAs triggered significantly higher levels of type I interferon compared to the m6A-sufficient viral RNAs, in a RIG-I-dependent manner. Reconstitution of the RIG-I pathway revealed that m6A-deficient virion RNA induced higher expression of RIG-I, bound to RIG-I more efficiently, enhanced RIG-I ubiquitination, and facilitated RIG-I conformational rearrangement and oligomerization. Furthermore, the m6A binding protein YTHDF2 is essential for suppression of the type I interferon signaling pathway, including by virion RNA. Collectively, our results suggest that several families in NNS RNA viruses acquire m6A in viral RNA as a common strategy to evade host innate immunity.IMPORTANCE The nonsegmented negative-sense (NNS) RNA viruses share many common replication and gene expression strategies. There are no vaccines or antiviral drugs for many of these viruses. We found that representative members of the families Pneumoviridae, Paramyxoviridae, and Rhabdoviridae among the NNS RNA viruses acquire m6A methylation in their genome and antigenome as a means to escape recognition by host innate immunity via a RIG-I-dependent signaling pathway. Viral RNA lacking m6A methylation induces a significantly higher type I interferon response than m6A-sufficient viral RNA. In addition to uncovering m6A methylation as a common mechanism for many NNS RNA viruses to evade host innate immunity, this study discovered a novel strategy to enhance type I interferon responses, which may have important applications in vaccine development, as robust innate immunity will likely promote the subsequent adaptive immunity.