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1.
Mol Immunol ; 139: 202-210, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34583098

RESUMO

A balance between the positive and negative regulation of toll-like receptor (TLR) signaling pathways is required to avoid detrimental and inappropriate inflammatory responses. Although some protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination have been demonstrated to potently modulate innate immune responses, the role of methylation, an important PTM, control of TLR4 signaling pathway remains unclear. In this study, we found that protein arginine methyltransferase 1, 2 and 3 (PRMT1, 2 and 3) were recruited to methylate TLR4-CD (cytoplasmic domain) after lipopolysaccharide (LPS) stimulation respectively, but the effect of PRMT2 on arginine methylation of TLR4-CD is the most significant among above three PRMTs, which prompted us to focus on PRMT2. Reduction of PRMT2 expression down-regulated arginine (R) methylation level of TLR4 with or without LPS treatment. Methionine 115 (M115) mediated PRMT2 catalyzed-arginine methylation of TLR4 on R731 and R812. Furthermore, PRMT1, 2 and 3 was recruited to methylate interferon regulatory factor 3 (IRF3) after LPS stimulation respectively, but the effect of PRMT2 on arginine methylation of IRF3 is the most significant among the above three PRMTs. Arginine methylation of TLR4 on R812 or arginine methylation of IRF3 on R285 mediated the interaction between TLR4 and IRF3 respectively. Arginine methylation of IRF3 on R285 induced by LPS led to its dimerization and promoted its translocation from the cytoplasm to the nucleus. In addition, the enhancement of arginine methylation of TLR4 induced by PRMT1 or 2 increased IRF3 transcription activity with or without LPS treatment, while PRMT2 with histidine 112 glutamine (H112Q) or methionine 115 isoleucine (M115I) mutation and TLR4 with arginine 812 lysine (R812K) mutation decreased it. Arginine methylation of TLR4 on R812 or PRMT2 enhanced interferon-ß (IFN-ß) production. Our study reveals a critical role for PRMT2 and protein arginine methylation in the enhancement of IFN-ß production via TLR4/IRF3 signaling pathway and may provide a therapeutic strategy to control endotoxemia.


Assuntos
Arginina/metabolismo , Regulação da Expressão Gênica/imunologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Endotoxemia/imunologia , Endotoxemia/metabolismo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Interferon beta/metabolismo , Metilação , Camundongos , Proteína-Arginina N-Metiltransferases/imunologia , Células RAW 264.7 , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
2.
Viruses ; 13(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34452305

RESUMO

A weak production of INF-ß along with an exacerbated release of pro-inflammatory cytokines have been reported during infection by the novel SARS-CoV-2 virus. SARS-CoV-2 encodes several proteins able to counteract the host immune system, which is believed to be one of the most important features contributing to the viral pathogenesis and development of a severe clinical picture. Previous reports have demonstrated that SARS-CoV-2 N protein, along with some non-structural and accessory proteins, efficiently suppresses INF-ß production by interacting with RIG-I, an important pattern recognition receptor (PRR) involved in the recognition of pathogen-derived molecules. In the present study, we better characterized the mechanism by which the SARS-CoV-2 N counteracts INF-ß secretion and affects RIG-I signaling pathways. In detail, when the N protein was ectopically expressed, we noted a marked decrease in TRIM25-mediated RIG-I activation. The capability of the N protein to bind to, and probably mask, TRIM25 could be the consequence of its antagonistic activity. Furthermore, this interaction occurred at the SPRY domain of TRIM25, harboring the RNA-binding activity necessary for TRIM25 self-activation. Here, we describe new findings regarding the interplay between SARS-CoV-2 and the IFN system, filling some gaps for a better understanding of the molecular mechanisms affecting the innate immune response in COVID-19.


Assuntos
COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteína DEAD-box 58/imunologia , Receptores Imunológicos/imunologia , SARS-CoV-2/imunologia , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , COVID-19/genética , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteína DEAD-box 58/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Regiões Promotoras Genéticas , Receptores Imunológicos/genética , SARS-CoV-2/genética , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética
3.
Front Immunol ; 12: 671471, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079553

RESUMO

Our previous studies reported that duck Tembusu virus nonstructural protein 2A (NS2A) is a major inhibitor of the IFNß signaling pathway through competitively binding to STING with TBK1, leading to a reduction in TBK1 phosphorylation. Duck TMUV NS2B3 could cleave and bind STING to subvert the IFNß signaling pathway. Here, we found that overexpression of duck TMUV NS4B could compete with TBK1 in binding to STING, reducing TBK1 phosphorylation and inhibiting the IFNß signaling pathway by using the Dual-Glo® Luciferase Assay System and the NanoBiT protein-protein interaction (PPI) assay. We further identified the E2, M3, G4, W5, K10 and D34 residues in NS4B that were important for its interaction with STING and its inhibition of IFNß induction, which were subsequently introduced into a duck TMUV replicon and an infectious cDNA clone. We found that the NS4B M3A mutant enhanced RNA replication and exhibited significantly higher titer levels than WT at 48-72 hpi but significantly decreased mortality (80%) in duck embryos compared to WT (100%); the NS4B G4A and R36A mutants slightly reduced RNA replication but exhibited the same titer levels as WT. However, the NS4B R36A mutant did not attenuate the virulence in duck embryos, whereas the G4A mutant significantly decreased the mortality (70%) of duck embryos. In addition, the NS4B W5A mutant did not affect viral replication, whereas the D34A mutant slightly reduced RNA replication, and both mutants exhibited significantly lower titer levels than the WT and significantly decreased mortality (90% and 70%, respectively) in duck embryos. Hence, our findings provide new insight into the development of attenuated flaviviruses by targeting the disabling viral strategies used to evade the innate defense mechanisms.


Assuntos
Doenças das Aves/imunologia , Patos/virologia , Infecções por Flavivirus/virologia , Flavivirus/patogenicidade , Interferon beta/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Patos/imunologia , Infecções por Flavivirus/imunologia , Virulência
4.
mBio ; 12(3): e0100521, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34125604

RESUMO

DEAD (Glu-Asp-Ala-Glu) box RNA helicases have been proven to contribute to antiviral innate immunity. The DDX21 RNA helicase was identified as a nuclear protein involved in rRNA processing and RNA unwinding. DDX21 was also proven to be the scaffold protein in the complex of DDX1-DDX21-DHX36, which senses double-strand RNA and initiates downstream innate immunity. Here, we identified that DDX21 undergoes caspase-dependent cleavage after virus infection and treatment with RNA/DNA ligands, especially for RNA virus and ligands. Caspase-3/6 cleaves DDX21 at D126 and promotes its translocation from the nucleus to the cytoplasm in response to virus infection. The cytoplasmic cleaved DDX21 negatively regulates the interferon beta (IFN-ß) signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. Thus, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus. IMPORTANCE Innate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu) box RNA helicases, originally considered to be involved in RNA processing and RNA unwinding, have been shown to play an important role in antiviral innate immunity. The precise regulation of innate immunity is critical for the host because the aberrant production of cytokines leads to unexpected pathological consequences. Here, we identified that DDX21 was cleaved at D126 by virus infection and treatment with RNA/DNA ligands via the caspase-3/6-dependent pathway. The cytoplasmic cleaved DDX21 negatively regulates the IFN-ß signaling pathway by suppressing the formation of the DDX1-DDX21-DHX36 complex. In sum, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immune response to virus.


Assuntos
Caspases/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Imunidade Inata , Viroses/imunologia , Células A549 , Caspases/classificação , Caspases/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Células HeLa , Humanos , Interferon beta/imunologia , Ligação Proteica , Transdução de Sinais/imunologia , Células THP-1
5.
Cell Physiol Biochem ; 55(3): 256-264, 2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-33984198

RESUMO

BACKGROUND/AIMS: During an immune response, type I interferon (IFN-I) signaling induces a wide range of changes, including those which are required to overcome viral infection and those which suppress cytotoxic T cells to avoid immunopathology. During certain bacterial infections, IFN-I signaling exerts largely detrimental effects. Although the IFN-I family of proteins all share one common receptor, biologic responses to signaling vary depending on IFN-I subtype. Here, we asked if one IFN-I subtype dominates the pro-bacterial effect of IFN-I signaling and found that control of Listeria monocytogenes (L.m.) infection is more strongly suppressed by IFN-ß than IFN-α. METHODS: To study this, we measured bacterial titers in IFNAR-/-, IFN-ß­/­, Stat2-/-, Usp18fl/fl and Usp18fl/fl x CD11c-Cre mice models in addition to IFN-I blocking antibodies. Moreover, we measured interferon stimulated genes in bone marrow derived dendritic cells after treatment with IFN-α4 and IFN-ß. RESULTS: Specifically, we show that genetic deletion of IFN-ß or antibody-mediated IFN-ß neutralization was sufficient to reduce bacterial titers to levels similar to those observed in mice that completely lack IFN-I signaling (IFNAR-/- mice). However, IFN-α blockade failed to significantly reduce L.m. titers, suggesting that IFN-ß is the dominant IFN-I subtype responsible for the pro-bacterial effect of IFN-I. Mechanistically, when focusing on IFN-I signals to dendritic cells, we found that IFN-ß induces ISGs more robustly than IFN-α, including USP18, the protein we previously identified as driving the pro-bacterial effects of IFN-I. Further, we found that this induction was STAT1/STAT2 heterodimer- or STAT2/STAT2 homodimer-dependent, as STAT2-deficient mice were more resistant to L.m. infection. CONCLUSION: In conclusion, IFN-Β is the principal member of the IFN-I family responsible for driving the pro-bacterial effect of IFN-I.


Assuntos
Interferon-alfa/imunologia , Interferon beta/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Feminino , Interferon-alfa/genética , Interferon beta/genética , Listeriose/genética , Masculino , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/imunologia
6.
J Immunol ; 206(10): 2420-2429, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33941660

RESUMO

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.


Assuntos
COVID-19/imunologia , Proteínas de Ligação ao GTP/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Transglutaminases/imunologia , Animais , COVID-19/genética , COVID-19/patologia , Proteínas de Ligação ao GTP/genética , Humanos , Fator Regulador 3 de Interferon/genética , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transglutaminases/genética
7.
J Infect ; 83(1): 61-68, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33892015

RESUMO

Caspases are classified as inflammatory or apoptotic category. Inflammatory caspases participate in inflammasome activation, while apoptotic caspases mediate apoptotic activation. Previous studies have shown that apoptotic caspases prevent the production of IFN-ß during apoptosis or virus infection. However, the relationship between apoptotic caspases and IFN-ß production during intracellular bacterial infection is still unclear. Here, we investigated the role of apoptotic caspases in IFN-ß production induced by Mycobacterium bovis (M. bovis) infection. M. bovis is an intracellular bacterium and belongs to the Mycobacterium tuberculosis complex. M. bovis infection can cause tuberculosis in animals and human beings. In the current study, we found that M. bovis infection triggered mitochondrial stress, which caused the leakage of cytochrome c into the cytoplasm, and in turn, activated the downstream caspase-9 and-3. Furthermore, our results showed that activation of apoptotic caspases reduced IFN-ß production during M. bovis infection and vice versa. Confocal microscopy analysis revealed that apoptotic caspases prevented IFN-ß production by decreasing p-IRF3 nuclear translocation. Our findings demonstrate that apoptotic caspases negatively regulate the production of IFN-ß induced by an intracellular bacterial infection.


Assuntos
Apoptose , Caspases , Interferon beta/imunologia , Macrófagos/imunologia , Mycobacterium bovis , Animais , Caspases/genética , Macrófagos/microbiologia , Camundongos , Tuberculose
9.
Vet Immunol Immunopathol ; 235: 110231, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33740613

RESUMO

This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.


Assuntos
Citocinas/genética , Interferon Tipo I/genética , Interferon beta/genética , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Ácido Ascórbico/farmacologia , Linhagem Celular , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/imunologia , Interferon beta/imunologia , Macrófagos/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Rutina/farmacologia , Suínos , alfa-Tocoferol/farmacologia
10.
Nat Commun ; 12(1): 1930, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772027

RESUMO

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.


Assuntos
Proteínas de Ligação a DNA/imunologia , Interferon beta/imunologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/imunologia , Proteínas de Ligação a RNA/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Imunoterapia/métodos , Interferon beta/metabolismo , Camundongos Endogâmicos C57BL , Mutação , Vírus Oncolíticos/metabolismo , Vírus Oncolíticos/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia
11.
Front Immunol ; 12: 573078, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33692778

RESUMO

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-ß promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-ß production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.


Assuntos
Alphacoronavirus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Interferon beta/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fator 3 Associado a Receptor de TNF/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alphacoronavirus/imunologia , Animais , Linhagem Celular , Coronavirus/imunologia , Coronavirus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/imunologia , Interferon beta/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Suínos , Fator 3 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo
12.
PLoS Pathog ; 17(2): e1008690, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33635931

RESUMO

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.


Assuntos
COVID-19/virologia , Grânulos Citoplasmáticos/metabolismo , Endorribonucleases/imunologia , Endorribonucleases/metabolismo , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , COVID-19/metabolismo , Linhagem Celular , Coronavirus/imunologia , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/virologia , Humanos , Interferon beta/imunologia , Interferon beta/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais , Replicação Viral/fisiologia
13.
Mol Cell ; 81(6): 1187-1199.e5, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33581076

RESUMO

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.


Assuntos
Fator de Iniciação 4E em Eucariotos/imunologia , Imunidade Inata , MicroRNAs/imunologia , Biossíntese de Proteínas/imunologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Animais , Fator de Iniciação 4E em Eucariotos/genética , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/imunologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Infecções por Vírus de RNA/genética , Vírus de RNA/genética
14.
J Clin Invest ; 131(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393505

RESUMO

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.


Assuntos
Córtex Cerebral/imunologia , Fibroblastos/imunologia , Herpesvirus Humano 1/imunologia , Interferon beta/imunologia , Neurônios/imunologia , Receptor 3 Toll-Like/imunologia , Vesiculovirus/imunologia , Animais , Linhagem Celular , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Fibroblastos/patologia , Fibroblastos/virologia , Humanos , Interferon beta/genética , Camundongos , Camundongos Knockout , Neurônios/patologia , Neurônios/virologia , Receptor 3 Toll-Like/genética
15.
Vet Microbiol ; 254: 108994, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33486326

RESUMO

Porcine epidemic diarrhea virus (PEDV) encodes many multifunctional proteins that inhibit host innate immune response during virus infection. As one of important structural proteins, PEDV E protein has been found to block the production of type I interferon (IFN) in virus life cycle, but little is known about this process that E protein subverts host innate immune. Thus, in this present study, we initiated the construction of eukaryotic expression vectors to express PEDV E protein. Subsequently, cellular localization analysis was performed and the results showed that the majority of PEDV E protein distributed at cytoplasm and localized in endoplasmic reticulum (ER). Over-expression of PEDV E protein significantly inhibited poly(I:C)-induced IFN-ß and IFN-stimulated genes (ISGs) productions. We also found that PEDV E protein remarkably suppressed the protein expression of RIG-I signaling-associated molecules, but all their corresponding mRNA levels remained unaffected and unchanged. Furthermore, PEDV E protein obviously interfered with the translocation of IRF3 from cytoplasm to nucleus through direct interaction with IRF3, which is crucial for the IFN-ß production induced by poly(I:C). Taken together, our results suggested that PEDV E protein acts as an IFN-ß antagonist through suppression of the RIG-I-mediated signaling. This study will pave the way for the further investigation into the molecular mechanisms by which PEDV E protein evades host innate immune response.


Assuntos
Proteína DEAD-box 58/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Interferon beta/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Proteínas Virais/genética , Animais , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/genética , Poli I-C/farmacologia , Vírus da Diarreia Epidêmica Suína/química , Vírus da Diarreia Epidêmica Suína/efeitos dos fármacos , Vírus da Diarreia Epidêmica Suína/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Suínos , Proteínas Virais/metabolismo
16.
Vet Microbiol ; 252: 108918, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33191000

RESUMO

Porcine haemagglutinating encephalomyelitis virus (PHEV) is a member of coronavirus that causes acute infectious disease and high mortality in piglets. The transcription factor IRF3 is a central regulator of type I interferon (IFN) innate immune signalling. Here, we report that PHEV infection of RAW264.7 cells results in strong suppression of IFN-ß production in the early stage. A comparative analysis of the upstream effector of IFN-ß transcription demonstrated that deactivation of IRF3, but not p65 or ATF-2 proteins, is uniquely attributed to failure of early IFN-ß induction. Moreover, the RIG-I/MDA5/MAVS/TBK1-dependent protective response that regulates the IRF3 pathway is not disrupted by PHEV and works well underlying the deactivated IRF3-mediated IFN-ß inhibition. After challenge with poly(I:C), a synthetic analogue of dsRNA used to stimulate IFN-ß secretion in the TLR-controlled pathway, we show that PHEV and poly(I:C) regulate IFN-ß-induction via two different pathways. Collectively, our findings reveal that deactivation of IRF3 is a specific mechanism that contributes to termination of type I IFN signalling during early infection with PHEV independent of the conserved RIG-I/MAVS/MDA5/TBK1-mediated innate immune response.


Assuntos
Betacoronavirus 1/imunologia , Infecções por Coronavirus/veterinária , Fator Regulador 3 de Interferon/genética , Interferon beta/imunologia , Animais , Betacoronavirus 1/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Camundongos , Poli I-C/farmacologia , Células RAW 264.7 , Transdução de Sinais/imunologia
17.
Curr Protein Pept Sci ; 22(1): 19-28, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33292152

RESUMO

Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.


Assuntos
Citocinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional , Ubiquitina Tiolesterase/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Citocinas/química , Citocinas/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Ubiquitina Tiolesterase/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/imunologia
18.
Pathog Dis ; 79(1)2021 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-33289808

RESUMO

A vast proportion of coronavirus disease 2019 (COVID-19) individuals remain asymptomatic and can shed severe acute respiratory syndrome (SARS-CoV) type 2 virus to transmit the infection, which also explains the exponential increase in the number of COVID-19 cases globally. Furthermore, the rate of recovery from clinical COVID-19 in certain pockets of the globe is surprisingly high. Based on published reports and available literature, here, we speculated a few immunovirological mechanisms as to why a vast majority of individuals remain asymptomatic similar to exotic animal (bats and pangolins) reservoirs that remain refractile to disease development despite carrying a huge load of diverse insidious viral species, and whether such evolutionary advantage would unveil therapeutic strategies against COVID-19 infection in humans. Understanding the unique mechanisms that exotic animal species employ to achieve viral control, as well as inflammatory regulation, appears to hold key clues to the development of therapeutic versatility against COVID-19.


Assuntos
COVID-19/imunologia , Síndrome da Liberação de Citocina/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Receptores KIR/imunologia , Receptores Semelhantes a Lectina de Células NK/imunologia , Zoonoses/imunologia , Animais , Animais Exóticos/virologia , Doenças Assintomáticas , COVID-19/genética , COVID-19/transmissão , COVID-19/virologia , Quirópteros/virologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/prevenção & controle , Síndrome da Liberação de Citocina/virologia , Reservatórios de Doenças , Eutérios/virologia , Expressão Gênica , Especificidade de Hospedeiro , Humanos , Tolerância Imunológica , Imunidade Inata , Interferon beta/deficiência , Interferon beta/genética , Interferon beta/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Monócitos/imunologia , Monócitos/virologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptores KIR/deficiência , Receptores KIR/genética , Receptores Semelhantes a Lectina de Células NK/deficiência , Receptores Semelhantes a Lectina de Células NK/genética , SARS-CoV-2/patogenicidade , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Zoonoses/genética , Zoonoses/transmissão , Zoonoses/virologia
19.
APMIS ; 129(3): 143-151, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33230840

RESUMO

The effect of rhinovirus on airway epithelium is very well described. However, its influence on the vascular endothelium is unknown. The current study assesses the effect of rhinovirus HRV16 on the antiviral and inflammatory response in the human vascular endothelial cells (ECs). HRV16 increased IFN-ß, RANTES, and IP-10 mRNA expression and protein release. HRV16 copy number in ECs reached maximal value 10 h after incubation. Increase in virus copies was accompanied by the enhancement of Toll- and RIG-I-like receptors: TLR3, RIG-I, and MDA5. Additionally, HRV16 increased OAS-1 and PKR mRNA expression, enzymes responsible for virus degradation and inhibition of replication. ICAM-1 blockade decreased HRV16 copy number in ECs and inhibited IFN-ß, RANTES, IP-10, OAS1, PKR, TLR3, RIG-I, and MDA5 mRNA expression increase upon subsequent induction with HRV16. The vascular endothelium may be infected by human rhinovirus and generate antiviral and inflammatory innate response. Results of the study indicate the possible involvement of the vascular endothelium in the immunopathology of rhinoviral airway infections.


Assuntos
Endotélio Vascular/imunologia , Infecções por Picornaviridae/imunologia , Rhinovirus/imunologia , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Endotélio Vascular/virologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Interferon beta/genética , Interferon beta/imunologia , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/virologia , Rhinovirus/genética , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia
20.
Virus Res ; 291: 198220, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33152381

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a main sensor used to detect microbial DNA in the cytoplasm, which subsequently induces the production of interferon (IFN) via the cGAS/STING/IRF3 signaling pathway, leading to an antiviral response. However, some viruses have evolved multiple strategies to escape this process. Pseudorabies virus (PRV) is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, which can cause serious damage to the porcine industry. Many herpesvirus components have been reported to counteract IFN production, whereas little is known of PRV. In the present study, we found that PRV glycoprotein E (gE) was involved in counteracting cGAS/STING-mediated IFN production. Ectopic expression of gE decreased cGAS/STING-mediated IFN-ß promoter activity and the level of mRNA expression. Moreover, gE targeted at or downstream of IRF3 was found to inhibit IFN-ß production. However, gE did not affect the phosphorylation, dimerization and nuclear translocation of IRF3. Furthermore, gE is located on the nuclear membrane and could subsequently degrade CREB-binding protein (CBP). MG132, a proteasome inhibitor, decreased CBP degradation and restored the IFN-ß production induced by gE. Finally, gE-deleted PRV induced a higher level of IFN-ß production and reduced CBP degradation compared to wild-type PRV. Together, these results demonstrate that PRV gE can inhibit cGAS/STING-mediated IFN-ß production by degrading CBP to interrupt the enhanced assembly of IRF3 and CBP.


Assuntos
Proteína de Ligação a CREB/metabolismo , Herpesvirus Suídeo 1/imunologia , Imunidade Inata , Interferon beta/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Proteína de Ligação a CREB/genética , Chlorocebus aethiops , Células HEK293 , Herpesvirus Suídeo 1/química , Herpesvirus Suídeo 1/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Fosforilação , Transdução de Sinais/imunologia , Suínos , Células Vero
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