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1.
Vet Microbiol ; 247: 108785, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32768229

RESUMO

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Interferons/antagonistas & inibidores , Animais , Linhagem Celular , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/imunologia , Interferons/imunologia , Intestinos/citologia , Intestinos/virologia , Rim/citologia , Rim/virologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-32656094

RESUMO

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-ß (IFN-ß) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-ß promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-ß production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-ß production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus/metabolismo , Quinase I-kappa B/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Coronavirus/genética , Coronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Quinase I-kappa B/imunologia , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/imunologia , Receptores do Ácido Retinoico/metabolismo , Vírus Sendai/imunologia , Vírus Sendai/metabolismo , Transdução de Sinais , Suínos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
3.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Fosfoproteínas , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
4.
Cancer Sci ; 111(5): 1692-1698, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32112659

RESUMO

Inactivated hemagglutinating virus of Japan envelope (HVJ-E) has an antitumor effect and tumor immunity. We undertook an open-label, phase I, dose-escalation study in patients with castration-resistant prostate cancer (CRPC) to determine the safety and efficacy of intratumoral and s.c. injection of HVJ-E (GEN0101). Patients with CRPC, who were resistant to or unable to receive standard of care, were included. GEN0101 was injected directly into the prostate and s.c. in two 28-day treatment cycles. The primary end-points were to evaluate the safety and tolerability of GEN0101 and determine its recommended dose. The secondary end-points were to analyze the antitumor effect and tumor immunity. Three patients received 30 000 mNAU GEN0101 and 6 received 60 000 mNAU. There was no dose-limiting toxicity, and the recommended dose of GEN0101 was defined as 60 000 mNAU. Radiographically, 1 patient had stable disease and 2 had progressive disease in the low-dose group, whereas 5 patients had stable disease and 1 had progressive disease in the high-dose group. Three patients in the high-dose group showed reduction in lymph node metastasis. Prostate-specific antigen increase rates in the high-dose group were suppressed more than those in the low-dose group. Natural killer cell activity was enhanced in 2 patients of the low-dose group and in 5 patients in the high-dose group. In conclusion, intratumoral and s.c. injections of GEN0101 were well-tolerated and feasible to use. The study is registered with the UMIN Clinical Trials Registry (no. UMIN000017092).


Assuntos
Terapia Viral Oncolítica , Neoplasias de Próstata Resistentes à Castração/terapia , Vírus Sendai/imunologia , Proteínas do Envelope Viral/imunologia , Idoso , Anticorpos Antivirais/sangue , Relação Dose-Resposta a Droga , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos , Humanos , Injeções , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/sangue , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Segurança
5.
J Biol Chem ; 295(6): 1575-1586, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31914403

RESUMO

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.


Assuntos
Imunidade Inata , Monócitos/imunologia , Proteína 1 com Domínio SAM e Domínio HD/imunologia , Núcleo Celular/imunologia , Humanos , Infecções por Respirovirus/imunologia , Proteína 1 com Domínio SAM e Domínio HD/análise , Vírus Sendai/imunologia , Células THP-1 , Células U937
6.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915282

RESUMO

The virus-induced signaling adaptor (VISA) complex plays a critical role in the innate immune response to RNA viruses. However, the mechanism of VISA complex formation remains unclear. Here, we demonstrate that thioredoxin 2 (TRX2) interacts with VISA at mitochondria both in vivo and in vitro Knockdown and knockout of TRX2 enhanced the formation of the VISA-associated complex, as well as virus-triggered activation of interferon regulatory factor 3 (IRF3) and transcription of the interferon beta 1 (IFNB1) gene. TRX2 inhibits the formation of VISA aggregates by repressing reactive oxygen species (ROS) production, thereby disrupting the assembly of the VISA complex. Furthermore, our data suggest that the C93 residue of TRX2 is essential for inhibition of VISA aggregation, whereas the C283 residue of VISA is required for VISA aggregation. Collectively, these findings uncover a novel mechanism of TRX2 that negatively regulates VISA complex formation.IMPORTANCE The VISA-associated complex plays pivotal roles in inducing type I interferons (IFNs) and eliciting the innate antiviral response. Many host proteins are identified as VISA-associated-complex proteins, but how VISA complex formation is regulated by host proteins remains enigmatic. We identified the TRX2 protein as an important regulator of VISA complex formation. Knockout of TRX2 increases virus- or poly(I·C)-triggered induction of type I IFNs at the VISA level. Mechanistically, TRX2 inhibits the production of ROS at its C93 site, which impairs VISA aggregates at its C283 site, and subsequently impedes the assembly of the VISA complex. Our findings suggest that TRX2 plays an important role in the regulation of VISA complex assembly.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Viral da Expressão Gênica , Imunidade Inata , Proteínas Mitocondriais/metabolismo , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Tiorredoxinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta-1a/metabolismo , Poli I-C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Células THP-1
7.
J Biol Chem ; 295(2): 444-457, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767682

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that suppress the expression of multiple genes and are involved in numerous biologic functions and disorders, including human diseases. Here, we report that two miRNAs, miR-302b and miR-372, target mitochondrial-mediated antiviral innate immunity by regulating mitochondrial dynamics and metabolic demand. Using human cell lines transfected with the synthetic analog of viral dsRNA, poly(I-C), or challenged with Sendai virus, we found that both miRNAs are up-regulated in the cells late after viral infection and ultimately terminate the production of type I interferons and inflammatory cytokines. We found that miR-302b and miR-372 are involved in dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and disrupt mitochondrial metabolism by attenuating solute carrier family 25 member 12 (SLC25A12), a member of the SLC25 family. Neutralizing the effects of the two miRNAs through specific inhibitors re-established the mitochondrial dynamics and the antiviral responses. We found that SLC25A12 contributes to regulating the antiviral response by inducing mitochondrial-related metabolite changes in the organelle. Structure-function analysis indicated that SLC25A12, as part of a prohibitin complex, associates with the mitochondrial antiviral-signaling protein in mitochondria, providing structural insight into the regulation of the mitochondrial-mediated antiviral response. Our results contribute to the understanding of how miRNAs modulate the innate immune response by altering mitochondrial dynamics and metabolic demand. Manipulating the activities of miR-302b and miR-372 may be a potential therapeutic approach to target RNA viruses.


Assuntos
MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Infecções por Respirovirus/metabolismo , Vírus Sendai/fisiologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , MicroRNAs/imunologia , Mitocôndrias/imunologia , Mitocôndrias/virologia , Proteínas de Transporte da Membrana Mitocondrial/imunologia , Membranas Mitocondriais/imunologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/virologia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Vírus Sendai/imunologia
8.
Virus Genes ; 55(4): 520-531, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31129785

RESUMO

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-ß production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-ß production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-ß production and transcription factor IRF3 activation, but did not block IFN-ß production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.


Assuntos
Coronavirus/imunologia , Proteína DEAD-box 58/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Proteínas do Nucleocapsídeo/imunologia , Suínos/virologia , Animais , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/antagonistas & inibidores , Interferon beta/genética , Ligação Proteica , RNA de Cadeia Dupla/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Vírus Sendai/imunologia , Doenças dos Suínos/virologia
9.
J Interferon Cytokine Res ; 39(6): 331-346, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31090472

RESUMO

RNA helicases play an important role in the response to microbial infection. Retinoic acid inducible gene-I (RIG-I) and members of the RIG-I-like receptor (RLR) family of helicases function as cytoplasmic pattern recognition receptors (PRRs) whose actions are essential for recognition of RNA viruses. RIG-I association with pathogen-associated molecular patterns (PAMPs) within viral RNA leads to its activation and signaling via the mitochondrial antiviral signaling (MAVS) adapter protein. This interaction mediates downstream signaling events that drive the innate immune response to virus infection. Here we identify the DEAH-box RNA helicase DHX15 as a RLR binding partner and signaling cofactor. In human cells, DHX15 is required for virus-induced RLR signaling of innate immune gene expression. Knockdown of DHX15 increased susceptibility to infection by RNA viruses of diverse genera, including Paramyxoviridae, Rhabdoviridae, and Picornaviridae. DHX15 associates with RIG-I caspase activation and recruitment domains (CARDs) through its amino terminus, in which the complex is recruited to MAVS on virus infection. Importantly, although DHX15 cannot substitute for RIG-I in innate immune signaling, DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation in response to viral RNA. Our results define DHX15 as a coreceptor required for RLR innate immune responses to control RNA virus infection.


Assuntos
RNA Helicases/imunologia , Infecções por Vírus de RNA/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Células Cultivadas , Células HEK293 , Humanos
10.
Nat Microbiol ; 4(11): 1872-1884, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30988430

RESUMO

Outbreaks of viral infections are a global health burden. Although type I interferon (IFN-I) exerts broad-spectrum antiviral effects, its antiviral efficacy in host cells is largely restricted by viruses. How the antiviral efficacy of IFN-I can be improved remains to be explored. Here, we identified the ADP-ribosyltransferase poly(ADP-ribose) polymerase family member 11 (PARP11) as a potent regulator of IFN-I antiviral efficacy. PARP11 does not restrict IFN-I production induced by vesicular stomatitis virus or Sendai virus but inhibits the strength of IFN-I-activated signalling. Mechanistically, PARP11 mono-ADP-ribosylates the ubiquitin E3 ligase ß-transducin repeat-containing protein (ß-TrCP). Mono-ADP-ribosylation of ß-TrCP promotes IFNα/ß receptor subunit 1 (IFNAR1) ubiquitination and degradation. Moreover, PARP11 expression is upregulated by virus infections, including vesicular stomatitis virus, herpes simplex virus-1 and influenza A virus, thus promoting ADP-ribosylation-mediated viral evasion. We further highlight the potential for repurposing clinical ADP-ribosylation inhibitors. We found that rucaparib can target PARP11 to stabilize IFNAR1 and therefore exhibits efficient enhancement of IFN-I signalling and the host antiviral response. Consequently, rucaparib renders mice more resistant to viral infection. Our study updates the understanding of how ß-TrCP regulates its substrates and may provide a druggable target for improving IFN antiviral efficacy.


Assuntos
Interferon Tipo I/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Receptor de Interferon alfa e beta/química , Receptor de Interferon alfa e beta/metabolismo , Viroses/imunologia , Proteínas Contendo Repetições de beta-Transducina/metabolismo , ADP-Ribosilação , Animais , Células Cultivadas , Chlorocebus aethiops , Modelos Animais de Doenças , Células HEK293 , Células Hep G2 , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Camundongos , Proteólise , Vírus Sendai/imunologia , Transdução de Sinais , Ubiquitinação , Células Vero , Vesiculovirus/imunologia , Viroses/tratamento farmacológico , Viroses/metabolismo
11.
Arch Virol ; 164(6): 1639-1646, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982935

RESUMO

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Doença Hemorrágica de Coelhos/imunologia , Rotavirus/imunologia , Vírus Sendai/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Imunoensaio/veterinária , Coelhos , Kit de Reagentes para Diagnóstico
12.
J Immunol ; 202(8): 2254-2265, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842273

RESUMO

The nonreceptor tyrosine kinase c-Abl plays important roles in T cell development and immune responses; however, the mechanism is poorly understood. IFN regulatory factor 3 (IRF3) is a key transcriptional regulator of type I IFN-dependent immune responses against DNA and RNA viruses. The data in this study show that IRF3 is physically associated with c-Abl in vivo and directly binds to c-Abl in vitro. IRF3 is phosphorylated by c-Abl and c-Abl-related kinase, Arg, mainly at Y292. The inhibitor AMN107 inhibits IFN-ß production induced by poly(dA:dT), poly(I:C), and Sendai virus in THP-1 and mouse bone marrow-derived macrophage cells. IRF3-induced transcription of IFN-ß is significantly reduced by the mutation of Y292 to F. Moreover, AMN107 suppresses gene expression of absent in melanoma 2 (AIM2) and subsequently reduces inflammasome activation induced by cytosolic bacteria, dsDNA, and DNA viruses. Consistent with this finding, Francisella tularensis subsp. holarctica live vaccine strain (Ft LVS), which is known as an activator of AIM2 inflammasome, induces death in significantly more C57BL/6 mice treated with the Abl inhibitor AMN107 or c-Abl/Arg small interfering RNA than in untreated mice. This study provides new insight into the function of c-Abl and Arg in regulating immune responses and AIM2 inflammasome activation, especially against Ft LVS infection.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Proteínas Proto-Oncogênicas c-abl/imunologia , Animais , Arginina/imunologia , Proteínas de Ligação a DNA/imunologia , Francisella/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Vírus Sendai/imunologia , Células THP-1
13.
Mol Cell ; 74(1): 19-31.e7, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30878284

RESUMO

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Caspases/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Viroses/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caspase 2/genética , Caspase 2/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Caspases/genética , Feminino , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Masculino , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/genética , Vírus Sendai/imunologia , Vírus Sendai/patogenicidade , Transdução de Sinais , Células THP-1 , Vírus Vaccinia/imunologia , Vírus Vaccinia/patogenicidade , Viroses/genética , Viroses/imunologia , Viroses/virologia
14.
J Immunol ; 202(8): 2332-2347, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30804041

RESUMO

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1f/f mice and conventional Stat1-/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1-/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.


Assuntos
Células Epiteliais/imunologia , Pneumopatias/imunologia , Infecções por Respirovirus/imunologia , Fator de Transcrição STAT1/imunologia , Vírus Sendai/imunologia , Animais , Doença Crônica , Células Epiteliais/virologia , Pneumopatias/genética , Pneumopatias/virologia , Camundongos , Camundongos Knockout , Infecções por Respirovirus/genética , Fator de Transcrição STAT1/genética
15.
Viruses ; 11(2)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30769920

RESUMO

RNA virus invasion induces a cytosolic RIG-I-like receptor (RLR) signaling pathway by promoting assembly of the Mitochondrial antiviral-signaling protein (MAVS) signalosome and triggers the rapid production of type I interferons (IFNs) and proinflammatory cytokines. During this process, the pivotal kinase TANK binding kinase 1 (TBK1) is recruited to the MAVS signalosome to transduce a robust innate antiviral immune response by phosphorylating transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor (NF)-κB and promoting their nuclear translocation. However, the molecular mechanisms underlying the negative regulation of TBK1 are largely unknown. In the present study, we found that THO complex subunit 7 homolog (THOC7) negatively regulated the cellular antiviral response by promoting the proteasomal degradation of TBK1. THOC7 overexpression potently inhibited Sendai virus- or polyI:C-induced IRF3 dimerization and phosphorylation and IFN-ß production. In contrast, THOC7 knockdown had the opposite effects. Moreover, we simulated a node-activated pathway to show that THOC7 regulated the RIG-I-like receptors (RLR)-/MAVS-dependent signaling cascade at the TBK1 level. Furthermore, THOC7 was involved in the MAVS signalosome and promoted TBK1 degradation by increasing its K48 ubiquitin-associated polyubiquitination. Together, these findings suggest that THOC7 negatively regulates type I IFN production by promoting TBK1 proteasomal degradation, thus improving our understanding of innate antiviral immune responses.


Assuntos
Imunidade Celular , Imunidade Inata , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vírus Sendai/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Células MCF-7 , Fosforilação , Complexo de Endopeptidases do Proteassoma/imunologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA/genética , Vírus Sendai/genética , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação
16.
Viral Immunol ; 31(10): 676-682, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30265587

RESUMO

microRNAs have been reported to play crucial roles in various biological processes, including cell proliferation, apoptosis, tumor genesis, and viral infections. miR-26b has been found to be involved in the pathogenesis of multiple tumors, however, little is known about the role it plays in innate immune responses. In this study, we report that miR-26b is able to induce type-I interferon (IFN) expression, which was supported by both quantitative real time polymerase chain reaction and luciferase reporter assays. Conversely, production of IFN was reduced upon inhibition of miR-26b. Sequentially, ectopic expression of miR-26b led to upregulated expression of STAT1 and IFN-stimulated genes (ISGs). Furthermore, overexpression of miR-26b repressed the replication of vesicular stomatitis virus (VSV) and Sendai virus (SeV). In turn, IFN was able to induce the expression of miR-26b in a time-dependent manner. In all, we found that miR-26b could inhibit VSV replication through upregulation of type-I IFNs and ISGs and could in turn be upregulated by IFNs.


Assuntos
Imunidade Inata , Interferon Tipo I/metabolismo , MicroRNAs/metabolismo , Vírus Sendai/imunologia , Transdução de Sinais , Vesiculovirus/imunologia , Replicação Viral , Perfilação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Fatores Imunológicos/biossíntese , Fatores Imunológicos/genética , Luciferases/análise , Luciferases/genética , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sendai/crescimento & desenvolvimento , Vesiculovirus/crescimento & desenvolvimento
17.
Semin Nephrol ; 38(5): 513-520, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30177023

RESUMO

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis in the world. IgAN is characterized by mesangial deposits of IgA1-containing immune complexes. IgA1 usually co-deposits with complement C3 and variable IgG and/or IgM. Exactly 50 years have passed since IgAN was described, however, the pathogenesis of disease onset and progression have not been fully clarified. Animal models can re-create the complex immunologic microenvironments that foster human autoimmunity and nephritis and provide access to tissue compartments not readily examined in patients. Thus, multiple models that may be helpful in studies of specific aspects of IgAN have been developed. A unique spontaneous animal model of IgAN, the ddY mouse, was reported in 1985. These mice show mild proteinuria and glomerular IgA deposits, with a highly variable incidence and degree of glomerular injury owing to a heterogeneous genetic background. Thus, we intercrossed an early onset group of ddY mice in which the development of IgAN resulted in the establishment of a novel 100% early onset-grouped ddY mouse model with increased levels of aberrantly glycosylated IgA and immune complexes. Although the molecular features of human IgA1 are different from rodent IgA, human IgA1 knock-in (α1KI)-CD89 transgenic mice, which express both human IgA1 and CD89, show circulating and mesangial deposits of IgA1-soluble CD89 complexes that result in kidney inflammation, hematuria, and proteinuria. In this review, we introduce several murine models of IgAN that can be useful tools for the analysis of multiple aspects of the pathogenesis of IgAN, which may aid in the assessment of approaches for the treatment of IgAN.


Assuntos
Antígenos CD/genética , Modelos Animais de Doenças , Glomerulonefrite por IGA/genética , Imunoglobulina A/genética , Camundongos , Receptores Fc/genética , Animais , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Galactose/metabolismo , Técnicas de Introdução de Genes , Glomerulonefrite por IGA/imunologia , Glicosilação , Hematúria/genética , Hematúria/imunologia , Humanos , Imunoglobulina A/metabolismo , Lactalbumina/imunologia , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/imunologia , Nefrite/genética , Nefrite/imunologia , Proteinúria/genética , Proteinúria/imunologia , Receptores Fc/imunologia , Vírus Sendai/imunologia , Receptor Toll-Like 9/imunologia , Tricotecenos/imunologia , Uteroglobina/genética
18.
Front Immunol ; 9: 1796, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123219

RESUMO

The kinds of vaccine-induced T cell responses that are beneficial for protection against Mycobacterium tuberculosis (Mtb) infection are not adequately defined. We had shown that a novel Sendai virus vectored vaccine, SeV85AB, was able to enhance immune protection induced by bacille Calmette-Guérin (BCG) in a prime-boost model. However, the profile of T cell responses boosted by SeV85AB was not determined. Herein, we show that the antigen-specific CD4+ and CD8+ T cell responses were both enhanced by the SeV85AB boost after BCG. Different profiles of antigen-specific po T cell subsets were induced in the local (lung) and systemic (spleen) sites. In the spleen, the CD4+ T cell responses that were enhanced by the SeV85AB boost were predominately IL-2 responses, whereas in the lung the greater increases were in IFN-γ- and TNF-α-producing CD4+ T cells; in CD8+ T cells, although IFN-γ was enhanced in both the spleen and lung, only IL-2+TNF-α+CD8+ T subset was boosted in the latter. After a challenge Mtb infection, there were significantly higher levels of recall IL-2 responses in T cells. In contrast, IFN-γ-producing cells were barely boosted by SeV85AB. After Mtb challenge a central memory phenotype of responding CD4+ T cells was a prominent feature in SeV85AB-boosted mice. Thus, our data strongly suggest that the enhanced immune protection induced by SeV85AB boosting was associated with establishment of an increased capacity to recall antigen-specific IL-2-mediated T cell responses and confirms this Sendai virus vector system as a promising candidate to be used in a heterologous prime-boost immunization regimen against TB.


Assuntos
Vacina BCG/imunologia , Vetores Genéticos , Imunização Secundária , Vírus Sendai , Subpopulações de Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Antígenos de Bactérias , Vacina BCG/administração & dosagem , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Imunidade Celular , Memória Imunológica , Pulmão/imunologia , Camundongos , Mycobacterium tuberculosis/imunologia , Vírus Sendai/genética , Vírus Sendai/imunologia , Baço/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas contra a Tuberculose/administração & dosagem
19.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021902

RESUMO

The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-ß) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-ß stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-ß. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-ß reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity.IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-ß transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.


Assuntos
Herpesvirus Humano 1/genética , Interferon beta/genética , Proteínas Serina-Treonina Quinases/genética , Vírus Sendai/genética , Vesiculovirus/genética , Fosfatases cdc25/genética , Células A549 , Ciclo Celular , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/imunologia , Fosforilação , Poli I-C/genética , Poli I-C/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Vírus Sendai/imunologia , Transdução de Sinais , Vesiculovirus/imunologia , Fosfatases cdc25/imunologia
20.
J Biol Chem ; 293(31): 11996-12010, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29903906

RESUMO

Chronic neuroinflammation is a characteristic of Parkinson's disease (PD). Previous investigations have shown that Parkin gene mutations are related to the early-onset recessive form of PD and isolated juvenile-onset PD. Further, Parkin plays important roles in mitochondrial quality control and cytokine-induced cell death. However, whether Parkin regulates other cellular events is still largely unknown. In this study, we performed overexpression and knockout experiments and found that Parkin negatively regulates antiviral immune responses against RNA and DNA viruses. Mechanistically, we show that Parkin interacts with tumor necrosis factor receptor-associated factor 3 (TRAF3) to regulate stability of TRAF3 protein by promoting Lys48-linked ubiquitination. Our findings suggest that Parkin plays a novel role in innate immune signaling by targeting TRAF3 for degradation and maintaining the balance of innate antiviral immunity.


Assuntos
Fibroblastos/imunologia , Imunidade Inata , Transdução de Sinais/imunologia , Fator 3 Associado a Receptor de TNF/genética , Ubiquitina-Proteína Ligases/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Chlorocebus aethiops , Fibroblastos/citologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Herpesvirus Humano 1/imunologia , Humanos , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Proteólise , Vírus Sendai/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Transdução Genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação , Células Vero , Vesiculovirus/imunologia
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