RESUMO
Cellular 5'-3' exoribonuclease 1 (XRN1) is best known for its role as a decay factor, which by degrading 5' monophosphate RNA after the decapping of DCP2 in P-bodies (PBs) in Drosophila, yeast, and mammals. XRN1 has been shown to degrade host antiviral mRNAs following the influenza A virus (IAV) PA-X-mediated exonucleolytic cleavage processes. However, the mechanistic details of how XRN1 facilitates influenza A virus replication remain unclear. In this study, we discovered that XRN1 and nonstructural protein 1 (NS1) of IAV are directly associated and colocalize in the PBs. Moreover, XRN1 downregulation impaired viral replication while the viral titers were significantly increased in cells overexpressing XRN1, which suggest that XRN1 is a positive regulator in IAV life cycle. We further demonstrated that the IAV growth curve could be suppressed by adenosine 3',5'-bisphosphate (pAp) treatment, an inhibitor of XRN1. In virus-infected XRN1 knockout cells, the phosphorylated interferon regulatory factor 3 (p-IRF3) protein, interferon beta (IFN-ß) mRNA, and interferon-stimulated genes (ISGs) were significantly increased, resulting in the enhancement of the host innate immune response and suppression of viral protein production. Our data suggest a novel mechanism by which the IAV hijacks the cellular XRN1 to suppress the host innate immune response and to facilitate viral replication. IMPORTANCE A novel mechanistic discovery reveals that the host decay factor XRN1 contributes to influenza A virus replication, which exploits XRN1 activity to inhibit RIG-I-mediated innate immune response. Here, we identified a novel interaction between viral NS1 and host XRN1. Knockdown and knockout of XRN1 expression in human cell lines significantly decreased virus replication while boosting RIG-I-mediated interferon immune response, suggesting that XRN1 facilitates influenza A virus replication. The pAp effect as XRN1 inhibitor was evaluated; we found that pAp was capable of suppressing viral growth. To our knowledge, this study shows for the first time that a negative-strand and nucleus-replicating RNA virus, as influenza A virus, can hijack cellular XRN1 to suppress the host RIG-I-dependent innate immune response. These findings provide new insights suggesting that host XRN1 plays a positive role in influenza A virus replication and that the inhibitor pAp may be used in novel antiviral drug development.
Assuntos
Exorribonucleases/genética , Exorribonucleases/imunologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Interferon beta/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Replicação Viral , Células A549 , Regulação para Baixo , Humanos , Imunidade Inata , Vírus da Influenza A/imunologia , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologiaRESUMO
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Interferon beta , Regiões Promotoras Genéticas , SARS-CoV-2 , RNA-Polimerase RNA-Dependente de Coronavírus/genética , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Interferon beta/biossíntese , Interferon beta/genética , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMO
Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes serious vomiting and diarrhea in piglets. Previous work demonstrated that PDCoV infection inhibits type I interferon (IFN) production. Here, we found that ectopic expression of PDCoV nsp10 significantly inhibited Sendai virus (SeV)-induced IFN-ß production by impairing the phosphorylation and nuclear translocation of two transcription factors, IRF3 and NF-κB p65 subunit. Interestingly, experiments with truncated mutants and site-directed mutagenesis revealed that PDCoV nsp10 mutants with missing or destroyed zinc fingers (ZFs) domains also impeded SeV-induced IFN-ß production, suggesting that nsp10 does not require its ZF domains to antagonize IFN-ß production. Further work found that co-expression of nsp10 with nsp14 or nsp16, two replicative enzymes, significantly enhanced the inhibitory effects of nsp10 on IFN-ß. Taken together, our results demonstrate that PDCoV nsp10 antagonizes IFN via a ZF-independent mechanism and has a synergistic effect with nsp14 and nsp16 on inhibiting IFN-ß production.
Assuntos
Deltacoronavirus/metabolismo , Interferon beta/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Mutação , Vírus Sendai/metabolismo , Transdução de Sinais , Suínos , Fator de Transcrição RelA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Dedos de ZincoRESUMO
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/metabolismoRESUMO
The open reading frame 8 (orf8) is an accessory protein of SARS-CoV-2. It has 121 amino acids with two genotypes, orf8L and orf8S. In this study, we overexpressed the orf8L and orf8S of SARS-CoV-2 as well as the orf8b of SARS-CoV to investigate their roles in the regulation of endoplasmic reticulum (ER) stress and the inhibition of interferon beta (IFNß) production. We found that the two genotypes of SARS-CoV-2 orf8 are capable of inducing ER stress without significant difference by triggering the activating transcription factor 6 (ATF6) and inositol-requiring enzymes 1 (IRE1) branches of the ER stress pathway. However, the third branch of ER stress pathway, i.e. the protein kinase-like ER kinase (PERK), was unaffected by the overexpression of SARS-CoV-2 orf8L or orf8S. Moreover, both orf8L and orf8S of SARS-CoV-2 are capable of down regulating the production of IFNß and interferon-stimulated genes (ISG), ISG15 and ISG56 induced by polyinosinic-polycytidylic acid (poly (I:C)). Moreover, we also found decreased nuclear translocation of Interferon regulatory factor 3 (IRF3), after overexpressing orf8L and orf8S induced by poly (I:C). Our data demonstrated that SARS-CoV-2 orf8 protein could induce ER stress by activating the ATF6 and IRE1 pathways, but not the PERK pathway, and functions as an interferon antagonist to inhibit the production of IFNß. However, these functions appeared not to be affected by the genotypes of SARS-CoV-2 orf8L and orf8S.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Evasão da Resposta Imune , Interferon beta/antagonistas & inibidores , Proteínas Virais/fisiologia , Fator 6 Ativador da Transcrição/fisiologia , Endorribonucleases/fisiologia , Células HEK293 , Humanos , Interferon beta/biossíntese , Proteínas Serina-Treonina Quinases/fisiologia , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas , Proteínas Virais/química , Proteína 1 de Ligação a X-Box/fisiologia , eIF-2 Quinase/fisiologiaRESUMO
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/metabolismoRESUMO
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 VeroRESUMO
This double-blind, randomized, placebo-controlled, dose-ascending, first-in-human study (NCT02766621) assessed the safety, tolerability, and pharmacokinetics (PK) of PF-06823859, an anti-interferon ß monoclonal antibody. Healthy subjects were randomized to single ascending doses (SADs) of intravenous PF-06823859 30, 100, 300, 900, or 2000 mg or placebo; to multiple ascending doses (MADs) of subcutaneous PF-06823859 100 or 300 mg or placebo (once every 2 weeks for a total of 3 doses); or to MAD of intravenous PF-06823859 600 mg or placebo (once every 3 weeks or once every 4 weeks for a total of 2 doses). The incidence, severity, and causal relationship of adverse events (AEs) were assessed, along with immunogenicity and PK. In total, 62 subjects were randomized to treatment (SAD, n = 35; MAD, n = 27). There were 76 treatment-emergent all-causality AEs in the SAD (PF-06823859: n = 25; placebo: n = 4) and MAD (PF-06823859: n = 40; placebo: n = 7) cohorts. In the SAD cohorts, all treatment-emergent all-causality AEs were mild in severity; 4 AEs of moderate severity were identified in the MAD cohorts. No dose-limiting AEs, serious AEs, treatment-related discontinuations, dose reductions, or deaths occurred. PF-06823859 exposure increased dose-proportionally, with half-life values ranging between 23 and 35 days. The estimated subcutaneous bioavailability was 43% to 44%. Immunogenicity incidence rates were low (antidrug antibodies, 12.5%; neutralizing antibodies, 2.1%). No immunogenically related clinical responses of concern were observed. In conclusion, PF-06823859 demonstrated an acceptable safety, tolerability, and PK profile that supports clinical development for treating disorders associated with increased interferon ß levels, such as dermatomyositis or systemic lupus erythematosus.
Assuntos
Anticorpos Monoclonais/farmacocinética , Doenças Autoimunes/tratamento farmacológico , Imunidade/efeitos dos fármacos , Interferon beta/antagonistas & inibidores , Administração Intravenosa , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Neutralizantes/efeitos dos fármacos , Doenças Autoimunes/imunologia , Disponibilidade Biológica , Estudos de Casos e Controles , Método Duplo-Cego , Tolerância a Medicamentos , Feminino , Meia-Vida , Voluntários Saudáveis , Humanos , Injeções Subcutâneas , Interferon beta/sangue , Interferon beta/metabolismo , Masculino , Pessoa de Meia-Idade , Farmacocinética , Placebos/administração & dosagem , SegurançaRESUMO
Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophora flavescens, suppresses experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), by inducing the production of immunomodulatory molecules, e.g., IL-10. In an effort to find the upstream pathway(s) of the mechanism underlying these effects, we have tested certain upregulated immunomodulatory molecules. Among them, we found increased levels of IL-27 and IFN-ß, one of the first-line MS therapies. Indeed, while low levels of IFN-ß production in sera and type I interferon receptor (IFNAR1) expression in spinal cord of saline-treated control EAE mice were detected, they were significantly increased after MAT treatment. Increased numbers of CD11b+IFN-ß+ microglia/infiltrating macrophages were observed in the CNS of MAT-treated mice. The key role of IFN-ß induction in the suppressive effect of MAT on EAE was further verified by administration of anti-IFN-ß neutralizing antibody, which largely reversed the therapeutic effect of MAT. Further, we found that, while MAT treatment induced production of IL-27 and IL-10 by CNS microglia/macrophages, this effect was significantly reduced by IFN-ß neutralizing antibody. Finally, the role of IFN-ß in MAT-induced IL-27 and IL-10 production was further confirmed in human monocytes in vitro. Together, our study demonstrates that MAT exerts its therapeutic effect in EAE through an IFN-ß/IL-27/IL-10 pathway, and is likely a novel, safe, low-cost, and effective therapy as an alternative to exogenous IFN-ß for MS.
Assuntos
Alcaloides/farmacologia , Autoimunidade/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Interferon beta/metabolismo , Quinolizinas/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental , Feminino , Imunofluorescência , Humanos , Interferon beta/antagonistas & inibidores , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Índice de Gravidade de Doença , MatrinasRESUMO
TRIM26, a member of the tripartite motif (TRIM) family has been shown to be involved in modulation of innate antiviral response. However, the functional characteristics of porcine TRIM26 (porTRIM26) are unclear. In this study, we used a synthesized antigen peptide to generate a polyclonal antibody against porTRIM26 with which to study the expression and function of porTRIM26. We demonstrated that polyinosinic:polycytidylic acid (poly (I:C)) stimulation and viral infection (vesicular stomatitis (VSV) or porcine reproductive and respiratory syndrome virus (PRRSV)) induce expression of porTRIM26, whereas knock-down expression of porTRIM26 promotes interferon (IFN)- production after poly (I:C) stimulation and virus infection (VSV or PRRSV). The importance of the porTRIM26-mediated modulation of the antiviral response was also shown in VSV- or PRRSV-infected cells. In summary, these findings show that porTRIM26 has an inhibitory role in IFN- expression and the antiviral response.
Assuntos
Antivirais/farmacologia , Interferon beta/antagonistas & inibidores , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas com Motivo Tripartido/metabolismo , Animais , Síndrome Respiratória e Reprodutiva Suína/tratamento farmacológico , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Suínos , Proteínas com Motivo Tripartido/genéticaRESUMO
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/imunologiaRESUMO
The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.
Assuntos
Interferon beta/antagonistas & inibidores , Genética Reversa , Vírus da Febre do Flebótomo Napolitano/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Células A549 , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Humanos , Insetos , Interferon beta/imunologia , Rim/citologia , Mutação , Vírus da Febre do Flebótomo Napolitano/imunologia , Células Vero , Proteínas Virais/genéticaRESUMO
Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly emerging enteric coronavirus, is considered to be associated with swine acute diarrhea syndrome (SADS) which has caused significantly economic losses to the porcine industry. Interactions between SADS-CoV and the host innate immune response is unclear yet. In this study, we used IPEC-J2 cells as a model to explore potential evasion strategies employed by SADS-CoV. Our results showed that SADS-CoV infection failed to induce IFN-ß production, and inhibited poly (I:C) and Sendai virus (SeV)-triggered IFN-ß expression. SADS-CoV also blocked poly (I:C)-induced phosphorylation and nuclear translocation of IRF-3 and NF-κB. Furthermore, SADS-CoV did not interfere with the activity of IFN-ß promoter stimulated by IRF3, TBK1 and IKKε, but counteracted its activation induced by IPS-1 and RIG-I. Collectively, this study is the first investigation that shows interactions between SADS-CoV and the host innate immunity, which provides information of the molecular mechanisms underlying SASD-CoV infection.
Assuntos
Alphacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Proteína DEAD-box 58/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Infecções por Coronavirus/virologia , Proteína DEAD-box 58/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , SuínosRESUMO
Porcine circovirus 3 (PCV3) is a novel circovirus that is associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multi-systemic inflammation. The type I Interferon (IFN) signaling pathway is an important innate immune signaling pathway for defense against viral infection. Many mammalian viruses inhibit host innate immune signaling through diverse strategies. Here, we found that the PCV3 capsid protein (Cap) significantly inhibited IFN-ß-stimulated response element (ISRE) promoter activity, suggesting that Cap suppresses IFN signaling. However, Cap did not affect expression and phosphorylation levels of STAT1 and STAT2 and did not interrupt the heterodimerization of pSTAT1 and pSTAT2. Although Cap interacted with KPAN1, it did not block the interaction between KPNA1 and pSTAT1 or the nuclear translocation of pSTAT1 and pSTAT2. Interestingly, we found that Cap inhibited the activation of ISRE promoter induced by IRF9-S2C. Mechanistically, Cap interacted with the transactivation domain of STAT2, a key protein in type I IFN signaling. In addition, we found that Cap bound to ISRE and prevented ISRE binding of IRF9-S2C. This work is the first to describe the mechanism of inhibition of IFN signaling by PCV3 Cap.
Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Interferon Tipo I/antagonistas & inibidores , Fator de Transcrição STAT2/imunologia , Transdução de Sinais/imunologia , Proteínas do Capsídeo/genética , Circovirus/classificação , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Interferon beta/antagonistas & inibidores , Interferon beta/imunologia , Fosforilação , Fator de Transcrição STAT1/imunologiaRESUMO
Chikungunya virus (CHIKV) is a single-stranded positive-sense RNA virus, belonging to the genus Alphavirus of the Togaviridae family. It causes multiple symptoms, including headache, fever, severe joint and muscle pain, and arthralgia. Since CHIKV was first isolated in Tanzania in 1952, there have been multiple outbreaks of chikungunya fever. However, its pathogenesis and mechanisms of viral immune evasion have been poorly understood. In addition, the exact roles of individual CHIKV genes on the host innate immune response remain largely unknown. To investigate if CHIKV-encoded genes modulate the type I interferon (IFN) response, each and every CHIKV gene was screened for its effects on the induction of the IFN-ß promoter. Here we report that CHIKV nsP2, E2 and E1 strongly suppressed activation of the IFN-ß promoter induced by the MDA5/RIG-I receptor signaling pathway, suggesting that nsP2, E2, and E1 are the major antagonists against induction of IFN-ß. Delineation of underlying mechanisms of CHIKV-mediated inhibition of the IFN-ß pathway may help develop virus-specific therapeutics and vaccines.
Assuntos
Vírus Chikungunya/metabolismo , Interferon beta/antagonistas & inibidores , Transdução de Sinais , Proteínas Virais/metabolismo , Vírus Chikungunya/genética , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Regiões Promotoras Genéticas , Receptores Imunológicos , Proteínas Virais/genéticaRESUMO
Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-ß (IFN-ß knockout [IFN-ß-KO] mice or mice treated with an IFN-ß-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-ß developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-ß-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-ß had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-ß-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-ß modulating neutrophil-mediated inflammation.IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-ß both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-ß protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.
Assuntos
Febre de Chikungunya/genética , Vírus Chikungunya/patogenicidade , Fator Regulador 7 de Interferon/genética , Interferon-alfa/genética , Interferon beta/genética , Neutrófilos/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Osso e Ossos/imunologia , Osso e Ossos/patologia , Osso e Ossos/virologia , Febre de Chikungunya/imunologia , Febre de Chikungunya/patologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Feminino , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Inflamação , Fator Regulador 7 de Interferon/deficiência , Fator Regulador 7 de Interferon/imunologia , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/deficiência , Interferon-alfa/imunologia , Interferon beta/antagonistas & inibidores , Interferon beta/deficiência , Interferon beta/imunologia , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Músculo Esquelético/virologia , Infiltração de Neutrófilos , Neutrófilos/patologia , Neutrófilos/virologia , Tarso Animal/imunologia , Tarso Animal/patologia , Tarso Animal/virologia , Replicação ViralRESUMO
Peste des petits ruminants virus (PPRV) is associated with global peste des petits ruminants resulting in severe economic loss. Peste des petits ruminants virus dampens host interferon-based signaling pathways through multiple mechanisms. Previous studies deciphered the role of V and C in abrogating IFN-ß production. Moreover, V protein directly interacted with signal transducers and activators of transcription 1 (STAT1) and STAT2 resulting in the impairment of host IFN responses. In our present study, PPRV infection inhibited both IFN-ß- and IFN-γ-induced activation of IFN-stimulated response element (ISRE) and IFN-γ-activated site (GAS) element, respectively. Both N and P proteins, functioning as novel IFN response antagonists, markedly suppressed IFN-ß-induced ISRE and IFN-γ-induced GAS promoter activation to impair downstream upregulation of various interferon-stimulated genes (ISGs) and prevent STAT1 nuclear translocation. Specifically, P protein interacted with STAT1 and subsequently inhibited STAT1 phosphorylation, whereas N protein neither interacted with STAT1 nor inhibited STAT1 phosphorylation as well as dimerization, suggesting that the N and P protein antagonistic effects were different. Though they differed in their relationship to STAT1, both proteins blocked JAK-STAT signaling, severely negating the host antiviral immune response. Our study revealed a new mechanism employed by PPRV to evade host innate immune response, providing a platform to study the interaction of paramyxoviruses and host response.
Assuntos
Interferons/antagonistas & inibidores , Nucleoproteínas/imunologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Fosfoproteínas/imunologia , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais/imunologia , Animais , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Interferon beta/antagonistas & inibidores , Interferon beta/imunologia , Interferon gama/antagonistas & inibidores , Interferon gama/imunologia , Interferons/imunologia , Janus Quinases/antagonistas & inibidores , Janus Quinases/imunologia , Proteínas do Nucleocapsídeo/imunologia , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/genética , Fator de Transcrição STAT1/imunologia , Replicação ViralRESUMO
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 , Receptores Imunológicos , Vírus Sendai/imunologia , Doenças dos Suínos/virologiaRESUMO
Sesamin, a representative sesame lignan, has health-promoting activities. Sesamin is converted into catechol derivatives and further into their glucuronides or sulfates in vivo, whereas the biological activities of sesamin metabolites remain unclear. We examined the inhibitory effects of sesamin metabolites on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophage-like J774.1 cells and found that a monocatechol derivative SC1, (7α,7'α,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane, has a much higher activity than sesamin and other metabolites. The inhibitory effects of SC1 glucuronides were time-dependently enhanced, associated with the intracellular accumulation of SC1 and the methylated form. SC1 glucuronides and SC1 attenuated the expression of inducible NO synthase (iNOS) and upstream interferon-ß (IFN-ß) in the LPS-stimulated macrophages. The inhibitory effects of SC1 glucuronides against NO production were canceled by the ß-glucuronidase inhibitor and enhanced by the catechol-O-methyltransferase inhibitor. Our results suggest that SC1 glucuronides exert the anti-inflammatory effects by inhibiting the IFN-ß/iNOS signaling through macrophage-mediated deconjugation.
Assuntos
Anti-Inflamatórios , Catecóis/farmacologia , Dioxóis/farmacologia , Glucuronídeos/farmacologia , Interferon beta/antagonistas & inibidores , Lignanas/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Animais , Catecol O-Metiltransferase/metabolismo , Catecóis/química , Catecóis/metabolismo , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Dioxóis/metabolismo , Glucuronidase/metabolismo , Glucuronídeos/química , Lignanas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Estrutura MolecularRESUMO
Proteasome is a large protein complex, which degrades most intracellular proteins. It regulates numerous cellular processes, including the removal of misfolded or unfolded proteins, cell cycle control, and regulation of apoptosis. However, the function of proteasome subunits in viral immunity has not been well characterized. In this study, we identified PSMB1, a member of the proteasome ß subunits (PSMB) family, as a negative regulator of innate immune responses during viral infection. Knockdown of PSMB1 enhanced the RNA virus-induced cytokine and chemokine production. Overexpression of PSMB1 abolished virus-induced activation of the interferon-stimulated response element (ISRE) and interferon beta (IFNß) promoters. Mechanistically, PSMB1 inhibited the activation of RIG-I-like receptor (RLR) and Toll-like receptor 3 (TLR3) signaling pathways. PSMB1 was induced after viral infection and its interaction with IKK-ε promoted degradation of IKK-ε through the ubiquitin-proteasome system. Collectively, our study demonstrates PSMB1 is an important regulator of innate immune signaling.