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1.
J Microbiol ; 57(12): 1126-1131, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31758397

RESUMO

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon ß (IFN-ß) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-ß expression would provide basis of the cell culture adaptation of HEV.


Assuntos
Vírus da Hepatite E/fisiologia , Helicase IFIH1 Induzida por Interferon/efeitos dos fármacos , Helicase IFIH1 Induzida por Interferon/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Técnicas de Cultura de Células , Proteína DEAD-box 58/efeitos dos fármacos , Proteína DEAD-box 58/metabolismo , Modelos Animais de Doenças , Células HEK293 , Hepatite E/virologia , Vírus da Hepatite E/enzimologia , Vírus da Hepatite E/patogenicidade , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação
2.
Vet Microbiol ; 238: 108430, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648727

RESUMO

Japanese Encephalitis Virus (JEV) is an important zoonotic flavivirus transmitted by mosquitos. JEV infection in sows primarily manifests as a reproductive disease such as abortion and transient infertility while in infected boars, it can cause orchitis. Previous studies mainly focused on the pathogenesis of human encephalitis caused by JEV infection, while few concentrations have been made to unveil the potential mechanism of reproductive dysfunction in JEV-infected pigs. In this study, histopathological analysis and immunohistochemistry staining was performed on testis of JEV-infected boars, indicating that JEV could infect testicular cells and cause inflammatory changes in testis. In vitro assays reveal that primary swine testicular cells and swine testis (ST) cells are highly permissive to JEV and significant inflammatory response was shown during JEV infection. Mechanically, we found that JEV infection increases the expression of retinoic acid-inducible gene I (RIG-I) and activates transcription factor NF-κB. Production of pro-inflammatory cytokines was greatly reduced in JEV infected testicular cells after knockout of RIG-I or treatment with the NF-κB specific inhibitor. In addition, activation of NF-κB was also significantly suppressed upon RIG-I knockout. Taken together, our results reveal that JEV could infect boar testicles, and RIG-I-NF-κB signaling pathway is involved in JEV-induced inflammation in swine testicular cells.


Assuntos
Proteína DEAD-box 58/metabolismo , Encefalite Japonesa/veterinária , NF-kappa B/metabolismo , Orquite/veterinária , Sus scrofa , Doenças dos Suínos/fisiopatologia , Animais , Células Cultivadas , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/complicações , Encefalite Japonesa/fisiopatologia , Técnicas In Vitro , Inflamação , Masculino , Orquite/etiologia , Transdução de Sinais/imunologia , Suínos , Doenças dos Suínos/virologia
3.
Adv Exp Med Biol ; 1172: 157-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628656

RESUMO

RIG-I-like receptors (RLRs) are an important family of pattern recognition receptors. They activate the immunological responses against viral infections by sensing RNAs in the cytoplasm. Recent studies provide significant insights into the activation and transduction mechanisms of RLRs family (members including RIG-I, MDA5, and LGP2). Here we review our current understanding of the structures of RLRs. Structural characterizations of RLRs family have revealed the mechanism of their actions at molecular level. The activation mechanisms of RIG-I and MDA5 are different, despite both of them have similar domain organizations and bind to dsRNA ligands. RIG-I, but not MDA5, adopts an auto-suppression conformation in the absence of RNA. In addition to ligand triggered receptor oligomerization, the activities of these receptors are also regulated by several posttranslational modifications, especially ubiquitination. Overall, these structural studies play critical roles in promoting the understanding of viral RNA recognition mechanisms by the host innate immune system, which also contribute to the designing of drugs for treatment of viral infection. However, much work remains to be done in studying the signaling pathway of MDA5 and LGP2, particularly by structural biology.


Assuntos
Proteína DEAD-box 58 , RNA Helicases DEAD-box , Imunidade Inata , RNA Viral , Animais , Proteína DEAD-box 58/química , Proteína DEAD-box 58/metabolismo , Humanos , Helicase IFIH1 Induzida por Interferon , RNA de Cadeia Dupla/metabolismo , RNA Viral/análise , RNA Viral/metabolismo , Transdução de Sinais , Viroses/imunologia
4.
Mol Cells ; 42(10): 721-728, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31600868

RESUMO

Non-structural protein 1 (NS1) of influenza virus has been shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with K d values of 1.62 ± 0.30 nM and 1.97 ± 0.27 nM, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteína DEAD-box 58/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas não Estruturais Virais/metabolismo , Animais , Células HEK293 , Humanos , Interferons/metabolismo , Camundongos , Ligação Proteica , Células RAW 264.7 , Replicação Viral
5.
PLoS Pathog ; 15(8): e1007983, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31433824

RESUMO

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and MDA5, initiates innate antiviral responses. Although regulation of RLR-mediated signal transduction has been extensively investigated, how the recognition of viral RNA by RLRs is regulated remains enigmatic. In this study, we identified heterogeneous nuclear ribonucleoprotein M (hnRNPM) as a negative regulator of RLR-mediated signaling. Overexpression of hnRNPM markedly inhibited RNA virus-triggered innate immune responses. Conversely, hnRNPM-deficiency increased viral RNA-triggered innate immune responses and inhibited replication of RNA viruses. Viral infection caused translocation of hnRNPM from the nucleus to the cytoplasm. hnRNPM interacted with RIG-I and MDA5, and impaired the binding of the RLRs to viral RNA, leading to inhibition of innate antiviral response. Our findings suggest that hnRNPM acts as an important decoy for excessive innate antiviral immune response.


Assuntos
Proteína DEAD-box 58/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , RNA Viral/metabolismo , Replicação Viral/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Humanos , Ligação Proteica , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/virologia , RNA Viral/genética , Transdução de Sinais
6.
J Microbiol ; 57(9): 803-811, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31452044

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a causative agent of severe-to-fatal pneumonia especially in patients with pre-existing conditions, such as smoking and chronic obstructive pulmonary disease (COPD). MERS-CoV transmission continues to be reported in the Saudi Arabian Peninsula since its discovery in 2012. However, it has rarely been epidemic outside the area except one large outbreak in South Korea in May 2015. The genome of the epidemic MERS-CoV isolated from a Korean patient revealed its homology to previously reported strains. MERS-CoV encodes 5 accessory proteins and generally, they do not participate in the genome transcription and replication but rather are involved in viral evasion of the host innate immune responses. Here we report that ORF8b, an accessory protein of MERS-CoV, strongly inhibits both MDA5- and RIG-I-mediated activation of interferon beta promoter activity while downstream signaling molecules were left largely unaffected. Of note, MDA5 protein levels were significantly down-regulated by ORF8b and co-expression of ORF4a and ORF4b. These novel findings will facilitate elucidation of mechanisms of virus-encoded evasion strategies, thus helping design rationale antiviral countermeasures against deadly MERS-CoV infection.


Assuntos
Infecções por Coronavirus/genética , Interferon beta/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Regiões Promotoras Genéticas , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Desenho de Drogas , Interações Hospedeiro-Patógeno , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Arábia Saudita , Vacinas Virais/genética , Vacinas Virais/imunologia
7.
Nat Commun ; 10(1): 3889, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467282

RESUMO

The innate response to a pathogen is critical in determining the outcome of the infection. However, the interplay of different cellular responses that are activated following viral infection and their contribution to innate antiviral signalling has not been clearly established. This work shows that flaviviruses, including Dengue, Zika, West Nile and Tick-borne encephalitis viruses, activate the unfolded protein response before transcription of interferon regulatory factor 3 induced genes. Infection in conditions of unfolded protein response priming leads to early activation of innate antiviral responses and cell intrinsic inhibition of viral replication, which is interferon regulatory factor 3 dependent. These results demonstrate that the unfolded protein response is not only a physiological reaction of the cell to viral infection, but also synergizes with pattern recognition sensing to mount a potent antiviral response.


Assuntos
Antivirais/farmacologia , Infecções por Flavivirus/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Imunidade Inata/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Proteína DEAD-box 58/metabolismo , Dengue/imunologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Encefalite Transmitida por Carrapatos/imunologia , Endorribonucleases/metabolismo , Feminino , Humanos , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Transcriptoma , Células Vero , Replicação Viral/efeitos dos fármacos , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/imunologia
8.
Eur J Pharmacol ; 860: 172543, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31323223

RESUMO

Ergosterol peroxide has been shown to exhibit anti-tumor, antioxidant and anti-bacterial properties. However, the effects of ergosterol peroxide isolated from the herbal Baphicacanthus cusia root on influenza virus infection remain poorly understood. In the present study, ergosterol peroxide (compound 22) was obtained from the B. cusia root and subjected to investigation regarding its immunoregulatory effect on influenza A virus (IAV)-induced inflammation in A549 human alveolar epithelial cells. The structure of compound 22 isolated from B. cusia root. was elucidated by NMR analyses. Structure determination showed that the chemical structure of compound 22 closely resembles that of ergosterol peroxide. We observed that ergosterol peroxide treatment significantly suppressed IAV-induced upregulation of RIG-I expression. Additionally, ergosterol peroxide inhibited the activation of RIG-I downstream signaling pathways, including p38 MAP kinase and NF-κB, which ultimately resulted in the reduced production of an array of pro-inflammatory mediators and interferons (IFN-ß and IFN-λ1). Interestingly, inhibitory effects of ergosterol peroxide on the expression of IFNs did not affect the expression of antiviral effectors or enhance viral replication. On the other hand, ergosterol peroxide effectively abolished the amplified production of pro-inflammatory mediators in cells pretreated with IFN-ß (500 ng/ml) prior to IAV infection. Moreover, Annexin V and Hoechst 33258 staining revealed that increased apoptosis of IAV-infected cells was reversed by the presence of ergosterol peroxide. Our findings suggest that ergosterol peroxide from the B. cusia root suppressed IAV-associated inflammation and apoptosis via blocking RIG-I signaling, which may serve as a supplementary approach to the treatment of influenza.


Assuntos
Apoptose/efeitos dos fármacos , Proteína DEAD-box 58/metabolismo , Ergosterol/análogos & derivados , Vírus da Influenza A Subtipo H1N1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células A549 , Animais , Cães , Ergosterol/química , Ergosterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Inflamação/virologia , Mediadores da Inflamação/metabolismo , Interferons/biossíntese , Células Madin Darby de Rim Canino
9.
EMBO J ; 38(14): e100978, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304625

RESUMO

Viral infection triggers the formation of mitochondrial antiviral signaling protein (MAVS) aggregates, which potently promote immune signaling. Autophagy plays an important role in controlling MAVS-mediated antiviral signaling; however, the exact molecular mechanism underlying the targeted autophagic degradation of MAVS remains unclear. Here, we investigated the mechanism by which RNF34 regulates immunity and mitophagy by targeting MAVS. RNF34 binds to MAVS in the mitochondrial compartment after viral infection and negatively regulates RIG-I-like receptor (RLR)-mediated antiviral immunity. Moreover, RNF34 catalyzes the K27-/K29-linked ubiquitination of MAVS at Lys 297, 311, 348, and 362 Arg, which serves as a recognition signal for NDP52-dependent autophagic degradation. Specifically, RNF34 initiates the K63- to K27-linked ubiquitination transition on MAVS primarily at Lys 311, which facilitates the autophagic degradation of MAVS upon RIG-I stimulation. Notably, RNF34 is required for the clearance of damaged mitochondria upon viral infection. Thus, we elucidated the mechanism by which RNF34-mediated autophagic degradation of MAVS regulates the innate immune response, mitochondrial homeostasis, and infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Viroses/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Lisina/metabolismo , Proteólise , Transdução de Sinais , Células THP-1 , Ubiquitinação , Viroses/metabolismo
10.
Vet Microbiol ; 233: 140-146, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176400

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25-RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon ß production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.


Assuntos
Proteína DEAD-box 58/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Ubiquitinação , Motivos de Aminoácidos , Animais , Linhagem Celular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas do Nucleocapsídeo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ligação Proteica , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Suínos , Transfecção , Replicação Viral
11.
Front Immunol ; 10: 176, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30814996

RESUMO

Scaffold proteins are defined as pivotal molecules that connect upstream receptors to specific effector molecules. Caspase recruitment domain protein 10 (CARD10) gene encodes a scaffold protein CARMA3, belongs to the family of CARD and membrane-associated guanylate kinase-like protein (CARMA). During the past decade, investigating the function of CARMA3 has revealed that it forms a complex with BCL10 and MALT1 to mediate different receptors-dependent signaling, including GPCR and EGFR, leading to activation of the transcription factor NF-κB. More recently, CARMA3 and its partners are also reported to be involved in antiviral innate immune response and DNA damage response. In this review, we summarize the biology of CARMA3 in multiple receptor-induced NF-κB signaling. Especially, we focus on discussing the function of CARMA3 in regulating NF-κB activation and antiviral IFN signaling in the context of recent progress in the field.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Proteínas Adaptadoras de Sinalização CARD/química , Proteína DEAD-box 58/metabolismo , Dano ao DNA , Receptores ErbB/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas-G/metabolismo
12.
Kidney Blood Press Res ; 44(1): 62-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30808838

RESUMO

BACKGROUND/AIMS: Dysregulation of interleukin-6 (IL-6) production in residual renal cells may play a pivotal role in the development of glomerulonephritis (GN). Given that Toll-like receptor 3 (TLR3) signaling has been implicated in the pathogenesis of some forms of GN, we examined activated TLR3-mediated IL-6 signaling in cultured normal human glomerular endothelial cells (GECs). METHODS: We treated GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of IL-6 and the cytosolic viral RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) using reverse transcription quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays. To further elucidate the effects of poly IC on this signaling pathway, we subjected the cells to small interfering RNA (siRNA) against TLR3, interferon (IFN)-ß, RIG-I, and MDA5. RESULTS: We found that poly IC induced the expression of RIG-I, MDA5 and IL-6 via TLR3/IFN-ß signaling in GECs. siRNA experiments revealed that both MDA5 and RIG-I were involved in the poly IC-induced expression of IL-6, with MDA5 being upstream of RIG-I. CONCLUSION: Interestingly, cytosolic sensors of viral RNA were found to be involved in IL-6 production via TLR3 signaling in GECs. Regional activation of TLR3/IFN-ß/ MDA5/RIG-I/IL-6 axis due to viral and "pseudoviral" infections is involved in innate immunity and inflammatory reactions in GECs. We believe this signaling pathway also plays a pivotal role in the development of some forms of GN.


Assuntos
Interleucina-6/biossíntese , Glomérulos Renais/citologia , Receptor 3 Toll-Like/metabolismo , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Células Endoteliais/metabolismo , Glomerulonefrite/etiologia , Humanos , Inflamação , Helicase IFIH1 Induzida por Interferon/metabolismo , Poli I-C/farmacologia , RNA Viral , Transdução de Sinais
13.
J Biol Chem ; 294(16): 6430-6438, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30804210

RESUMO

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.


Assuntos
Proteína DEAD-box 58 , DNA Helicases , Interferon beta , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose , Biossíntese de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , RNA de Cadeia Dupla , RNA Viral , Infecções por Rhabdoviridae , Vesiculovirus , Animais , Células COS , Proteína DEAD-box 58/química , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Ligação Proteica , Células RAW 264.7 , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/metabolismo , Transdução de Sinais/genética , Vesiculovirus/química , Vesiculovirus/genética , Vesiculovirus/metabolismo
14.
Trends Pharmacol Sci ; 40(2): 116-127, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30606502

RESUMO

RIG-I and MDA5 receptors are key sensors of pathogen-associated molecular pattern (PAMP)-containing viral RNA and transduce downstream signals to activate an antiviral and immunomodulatory response. Fifteen years of research have put them at the center of an ongoing hunt for novel pharmacological pan-antivirals, vaccine adjuvants, and antitumor strategies. Current knowledge testifies to the redundant, but also distinct, functions mediated by RIG-I and MDA5, opening opportunities for the use of specific and potent nucleic acid agonists. We critically discuss the evidence and remaining knowledge gaps that have an impact on the choice and design of optimal RNA ligands to achieve an appropriate immunostimulatory response, with limited adverse effects, for prophylactic and therapeutic interventions against viruses and cancer in humans.


Assuntos
Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Antivirais/farmacologia , Doenças Autoimunes/tratamento farmacológico , Proteína DEAD-box 58/química , Proteína DEAD-box 58/imunologia , Humanos , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/imunologia , Ligantes , Terapia de Alvo Molecular , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo
15.
Fish Shellfish Immunol ; 86: 1058-1063, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30593899

RESUMO

In mammals, virus infection of host cells triggers innate immune response, characterized by induction of interferon (IFN) and downstream IFN-stimulated genes (ISGs). The initiation of IFN antiviral response is dependent on host recognition of virus infection. In fish, similar IFN antiviral response is induced in response to RNA or DNA virus infection; however, the detailed mechanisms underlying recognition of a given virus and activation of downstream signaling remain largely unexplored. Using an infection model with Epithelioma papulosum cyprini (EPC) cells and spring viremia of carp virus (SVCV), a negative sense single-stranded RNA virus, we reported that fish RLR signaling pathway was involved in SVCV-triggered fish IFN response. IFN response was significantly initiated in EPC cells when infected with SVCV, as evidenced by activation of fish IFN promoters, upregulation of IFN and ISGs at mRNA and protein levels. However, function blockade of RIG-I and MDA5, two cytosolic receptors of fish RLR family, significantly attenuated the activation of fish IFN promoters and also the induction of fish IFN and ISGs by SVCV infection. Consistently, SVCV infection-triggered IFN response were blocked in EPC cells when transfected with the dominant negative mutants of pivotal RLR signaling factors, including MAVS, MITA, TBK1, IRF3 and IRF7. These results together shed light on the conservation of RLR-mediated IFN signaling that contributes to fish cells responding to RNA virus infection.


Assuntos
Doenças dos Peixes/imunologia , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Cyprinidae/imunologia , Proteína DEAD-box 58/metabolismo , Doenças dos Peixes/virologia , Imunidade Inata , Interferons/imunologia , Regiões Promotoras Genéticas , Infecções por Rhabdoviridae/imunologia
16.
Biochem Biophys Res Commun ; 508(3): 667-674, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30527812

RESUMO

Virus-induced signaling adaptor (VISA), which mediates the production of type I interferon, is crucial for the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. Upon viral infection, RIG-I recognizes double-stranded viral RNA and interacts with VISA to mediate antiviral innate immunity. However, the mechanisms underlying RIG/VISA-mediated antiviral regulation remain unclear. In this study, we confirmed that receptor for activated C kinase 1 (RACK1) interacts with VISA and attenuates the RIG/VISA-mediated antiviral innate immune signaling pathway. Overexpression of RACK1 inhibited the interferon-ß (IFN-ß) promoter; interferon-stimulated response element (ISRE); nuclear factor kappa B (NF-κB) activation; and dimerization of interferon regulatory factor 3 (IRF3) mediated by RIG-I, VISA, and TANK-binding kinase 1 (TBK1). A reduction in RACK1 expression level upon small interfering RNA knockdown increased RIG/VISA-mediated antiviral transduction. Additionally, RACK1 disrupted formation of the VISA-tumor necrosis factor receptor-associated factor 2 (TRAF2), VISA-TRAF3, and VISA-TRAF6 complexes during RIG-I/VISA-mediated signal transduction. Additionally, RACK1 enhanced K48-linked ubiquitination of VISA, attenuated its K63-linked ubiquitination, and decreased VISA-mediated antiviral signal transduction. Together, these results indicate that RACK1 interacts with VISA to repress downstream signaling and downregulates virus-induced IFN-ß production in the RIG-I/VISA signaling pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais/metabolismo , Proteína DEAD-box 58/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Quinase C Ativada/metabolismo , Transdução de Sinais , Técnicas de Silenciamento de Genes , Humanos , Interferon beta/biossíntese , Interferon beta/metabolismo , Lisina/metabolismo , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Vírus Sendai/fisiologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitinação
17.
Int J Cancer ; 144(7): 1645-1656, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30230526

RESUMO

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy.


Assuntos
Proteína DEAD-box 58/metabolismo , Células Matadoras Naturais/citologia , Melanoma/imunologia , Melanoma/terapia , RNA/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose , Técnicas de Cocultura , Citotoxicidade Imunológica , Humanos , Células Matadoras Naturais/imunologia , Ligantes , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , RNA/genética , Transfecção , Transplante Autólogo , Células Tumorais Cultivadas
18.
Eur J Immunol ; 49(1): 42-53, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30466171

RESUMO

Retinoic acid-inducible gene I (RIG-I) is a critical RNA virus sensor that initiates antiviral immune response through K63-linked ubiquitination. In this study, we demonstrated USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by directly deubiquitinating K63-linked RIG-I. USP14 knockdown significantly enhanced RIG-I-triggered type I IFN signaling and inhibited vesicular stomatitis virus (VSV) replication both in mouse peritoneal macrophages and THP1 cells. USP14 overexpression in HeLa cells attenuated RIG-I-triggered IFN-ß expression and promoted VSV replication. Besides, USP14-specific inhibitor, IU1, increased RIG-I-mediated type I IFN production and antiviral responses in vitro and in vivo. In addition, USP14 could interact with RIG-I and remove RIG-I K63-linked polyubiquitination chains. This article is the first to report that USP14 acts as a negative regulator in antiviral response through deubiquitinating K63-linked RIG-I. These findings provide insights into a potential new therapy targeting USP14 for RNA virus-related diseases.


Assuntos
Macrófagos/imunologia , Infecções por Rhabdoviridae/imunologia , Ubiquitina Tiolesterase/metabolismo , Vesiculovirus/fisiologia , Animais , Proteína DEAD-box 58/metabolismo , Feminino , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Transdução de Sinais , Células THP-1 , Ubiquitina Tiolesterase/genética , Ubiquitinação , Replicação Viral
19.
Fish Shellfish Immunol ; 84: 857-864, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30385247

RESUMO

Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state.


Assuntos
Cyprinidae/genética , Cyprinidae/imunologia , Proteína DEAD-box 58/genética , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Interferons/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia , Animais , Linhagem Celular , Proteína DEAD-box 58/metabolismo , Retroalimentação Fisiológica , Proteínas de Peixes/genética , Proteínas de Peixes/fisiologia , Interferons/metabolismo , Novirhabdovirus/fisiologia , Filogenia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética
20.
Dev Comp Immunol ; 90: 157-164, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30253130

RESUMO

RIG-I like receptor (RLR) signaling functions importantly in host innate immune response against RNA virus, which is tightly regulated by a number of mechanisms to prevent aberrant interferon production. The suppressor of IKKε (SIKE) has been identified as a suppressor of IKKε and TBK1, which are key components of RLR signaling. In this study, SIKE homologue (bcSIKE) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcSIKE varied in host cells in response to the stimulation of LPS, poly (I:C) and viruses. bcSIKE migrated around 27 KDa in immunoblot assay and distributed in both cytoplasm and nucleus of host cell in immunofluorescent (IF) staining test. bcSIKE showed no IFN-inducing ability in reporter assay and EPC cells expressing bcSIKE showed no enhanced antiviral ability against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV). However, bcSIKE obviously dampened the IFN-inducing ability of RLR signaling members in reporter assay when bcSIKE was co-expressed with these molecules in EPC cells. The association between bcSIKE and bcTBK1 has been identified through IF and co-immunoprecipitation (co-IP) assay. The plaque assay demonstrated clearly that bcTBK1-mediated antiviral activity in EPC cells against both GCRV and SVCV was down regulated by bcSIKE. All the data generated in this paper support the conclusion that bcSIKE interacts with bcTBK1 and inhibits bcTBK1-mediated antiviral signaling during host innate immune activation, which is reported in teleost for the first time.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus de RNA/fisiologia , Infecções por Reoviridae/imunologia , Reoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Rhabdoviridae/fisiologia , Animais , Células Cultivadas , Clonagem Molecular , Proteína DEAD-box 58/metabolismo , Proteínas de Peixes/metabolismo , Humanos , Imunidade Inata , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais
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