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1.
J Immunol ; 207(7): 1903-1910, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34497149

RESUMO

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I (encoded by Ddx58) and melanoma differentiation-associated gene 5 (MDA5) (encoded by Ifih1), are crucial for initiating antiviral responses. Endogenous retroviral elements (ERVs) are transposable elements derived from exogenous retroviruses that are integrated into the genome. KRAB-associated protein 1 (KAP1) is a key epigenetic suppressor of ERVs that protects cells from detrimental genome instability. Increased ERV transcripts are sensed by RLRs and trigger innate immune signaling. However, whether KAP1 directly controls RLRs activity remains unclear. In this study, we show that KAP1 attenuates RNA viral infection-induced type I IFNs and facilitates viral replication by inhibiting RIG-I/MDA5 expression in primary peritoneal macrophages (PMs) of C57BL/6J mice. Kap1 deficiency increases IFN-ß expression and inhibits vesicular stomatitis virus replication in C57BL/6J mice in vivo. Mechanistically, KAP1 binds to the promoter regions of Ddx58 and Ifih1 and promotes the establishment of repressive histone marks in primary PMs of C57BL/6J mice. Concordantly, KAP1 suppresses the expression of RIG-I and MDA5 at the transcriptional level in primary PMs of C57BL/6J mice. Our results establish that KAP1 epigenetically suppresses host antiviral responses by directly targeting RIG-1 and MDA5, thus facilitating the immune escape of RNA viruses.


Assuntos
Interferon beta , RNA , Animais , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , Epigênese Genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
2.
PLoS Pathog ; 17(8): e1009800, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34437657

RESUMO

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.


Assuntos
Interferon beta/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/farmacologia , SARS-CoV-2/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Células Vero , Proteínas Virais/genética
3.
J Immunol ; 207(5): 1411-1418, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34348973

RESUMO

The receptor for activated C kinase 1 (RACK1) adaptor protein has been implicated in viral infection. However, whether RACK1 promotes in vivo viral infection in mammals remains unknown. Moreover, it remains elusive how RACK1 is engaged in antiviral innate immune signaling. In this study, we report that myeloid RACK1 deficiency does not affect the development and survival of myeloid cells under resting conditions but renders mice less susceptible to viral infection. RACK1-deficient macrophages produce more IFN-α and IFN-ß in response to both RNA and DNA virus infection. In line with this, RACK1 suppresses transcriptional activation of type 1 IFN gene promoters in response to virus infection. Analysis of virus-mediated signaling indicates that RACK1 inhibits the phosphorylation of IRF3/7. Indeed, RACK1 interacts with IRF3/7, which is enhanced after virus infection. Further exploration indicates that virus infection triggers AMPK activation, which in turn phosphorylates RACK1 at Thr50 RACK1 phosphorylation at Thr50 enhances its interaction with IRF3/7 and thereby limits IRF3/7 phosphorylation. Thus, our results confirm that myeloid RACK1 promotes in vivo viral infection and provide insight into the control of type 1 IFN production in response to virus infection.


Assuntos
Proteínas Quinases Ativadas por AMP , Fator Regulador 3 de Interferon , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Camundongos , Fosforilação , Receptores de Quinase C Ativada , Transdução de Sinais
4.
J Virol ; 95(19): e0081521, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287046

RESUMO

Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae, harbors an accessory protein, named C protein, which facilitates viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles by binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, small interfering RNA (siRNA)-mediated gene knockdown studies suggested that neither Alix nor C protein is related to SeV budding. In the present study, we determined the crystal structure of a complex comprising the C-terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å to investigate the role of the complex in SeV budding. The structure revealed that a novel consensus sequence, LXXW, which is conserved among Respirovirus C proteins, is important for Alix binding. SeV possessing a mutated C protein with reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h postinfection compared with nonmutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, for binding to Alix. All together, these results highlight the critical role of C protein in SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been studied extensively to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating viral reproduction, but its role in budding remains controversial. In the present study, the crystal structure of the C-terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutant C proteins with different binding affinities to Alix and showed that the interaction between C and Alix is vital for viral budding. These findings provide new insights into the development of new antiviral drugs against hPIV1.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vírus Sendai/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Liberação de Vírus , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Cristalografia por Raios X , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Vírus Sendai/química , Vírus Sendai/genética , Vírus Sendai/metabolismo , Transdução de Sinais , Vírion/fisiologia
5.
J Exp Med ; 218(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34297037

RESUMO

The three classes of interferons (IFNs) share the ability to inhibit viral replication, activating cell transcriptional programs that regulate both innate and adaptive responses to viral and intracellular bacterial challenge. Due to their unique potency in regulating viral replication, and their association with numerous autoimmune diseases, the tightly orchestrated transcriptional regulation of IFNs has long been a subject of intense investigation. The protective role of early robust IFN responses in the context of infection with SARS-CoV-2 has further underscored the relevance of these pathways. In this viewpoint, rather than focusing on the downstream effects of IFN signaling (which have been extensively reviewed elsewhere), we will summarize the historical and current understanding of the stepwise assembly and function of factors that regulate IFNß enhancer activity (the "enhanceosome") and highlight opportunities for deeper understanding of the transcriptional control of the ifnb gene.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Interferon beta/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Elementos Facilitadores Genéticos , Interações Hospedeiro-Patógeno/genética , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Interferon beta/metabolismo , Regiões Promotoras Genéticas , SARS-CoV-2/patogenicidade , Transcrição Genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Mol Immunol ; 136: 110-117, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34098343

RESUMO

Reticuloendothelial virus (REV) is widely found in many domestic poultry areas and results in severe immunosuppression of infected chickens. This increases the susceptibility to other pathogens, which causes economic losses to the poultry industry. The aim of our study was to determine whether polyinosinic-polycytidylic acid [Poly (I: C)] treatment could inhibit REV replication in chicken macrophage-like cell line, HD11. We found that Poly (I: C) treatment could markedly inhibit REV replication in HD11 from 24 to 48 h post infection (hpi). Additionally, Poly (I: C) treatment could switch HD11 from an inactive type into M1-like polarization from 24 to 48 hpi. Furthermore, Poly (I: C) treatment promoted interferon-ß secretion from HD11 post REV infection. Moreover, Toll-like receptor-3 (TLR-3) mRNA and protein levels in HD11 treated with Poly (I: C) were markedly increased compared to those of HD11 not treated with Poly (I: C). The above results suggested that Poly (I: C) treatment switches HD11 into M1-like polarization to secret more interferon-ß and activate TLR-3 signaling, which contributes to block REV replication. Our findings provide a theoretical reference for further studying the underlying pathogenic mechanism of REV and Poly (I: C) as a potential therapeutic intervention against REV infection.


Assuntos
Antivirais/farmacologia , Indutores de Interferon/farmacologia , Poli I-C/farmacologia , Vírus da Reticuloendoteliose Aviária/crescimento & desenvolvimento , Receptor 3 Toll-Like/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Galinhas , Interferon beta/biossíntese , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Vírus da Reticuloendoteliose Aviária/efeitos dos fármacos , Infecções por Retroviridae/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Infecções Tumorais por Vírus/tratamento farmacológico
7.
Nucleic Acids Res ; 49(12): 6673-6686, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139015

RESUMO

G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.


Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Citostáticos/farmacologia , Quadruplex G/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular , Feminino , Compostos de Anéis Fundidos/farmacologia , Humanos , Imunidade Inata/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Células MCF-7 , Melanoma Experimental/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Micronúcleos com Defeito Cromossômico , Nucleotidiltransferases/metabolismo , Ativação Transcricional
8.
Vet Microbiol ; 258: 109098, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33984793

RESUMO

Porcine circovirus 2 (PCV2) has been proved to increase the risk of other pathogens infection via immunosuppression. Although the co-infection of PCV2 and porcine parvovirus (PPV) is commonly observed in worldwide, the relative immune mechanisms promoting PPV infection in PCV2-infected piglets are currently unknown. Herein, we found that PCV2 infection suppressed IFN-ß expression and promoted PPV infection in the piglets. Consistent with this finding, we confirmed that PCV2 infection significantly inhibited the induction of IFN-ß to promote PPV replication in cell level. Furthermore, PCV2 infection attenuated the K63-linked ubiquitination of STING induced by PPV, blocked the formation of complex of STING, TBK1 and IRF3, and further prevented the phosphorylation of TBK1 and IRF3, resulting in a decreased IFN-ß transcription response to PPV infection. Consistently, using cGAMP to direct stimulate STING also appeared a reduced STING-K63 ubiquitination and IFN-ß induction in PCV2-infected cells. However, we noted that knockdown of p38-MAPK signaling could markedly attenuate the inhibitory effect of PCV2 on STING-K63 ubiquitination, and improve the induction of IFN-ß in PCV2-infected whenever theses cells were challenged with PPV infection or cGAMP stimulation. Meanwhile, we found that PCV2 infection promoted the phosphorylation of USP21 to inhibit the K63 ubiquitination of STING and the transcription of IFN-ß via activation of p38-MAPK signaling. Taken together, our results demonstrate that PCV2 infection activates the p38-MAPK signaling pathway-mediated USP21 phosphorylation to inhibit the K63 ubiquitination of STING, which prevents the phosphorylation and transportation to the nucleus of IRF3, leading to an increase risk for PPV infection.


Assuntos
Circovirus/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Fator Regulador 3 de Interferon/genética , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Suínos , Testículo/citologia , Ubiquitinação
9.
Vet Microbiol ; 258: 109104, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34004569

RESUMO

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease.


Assuntos
Herpesvirus Suídeo 1/genética , Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/virologia , Doenças dos Suínos/prevenção & controle , Animais , Linhagem Celular , Interferon beta/genética , Interferon beta/metabolismo , Pseudorraiva/imunologia , Testes Sorológicos , Suínos , Doenças dos Suínos/virologia
10.
Biochem Biophys Res Commun ; 561: 52-58, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34020141

RESUMO

This is the first study to clone duck CCCH-type zinc finger antiviral protein (duZAP) from Jingjiang duck (Anas platyrhynchos). Full-length duZAP cDNA was 2154 bp and encoded a 717-amino acid polypeptide containing four highly conserved CCCH-type finger motifs, a WWE domain and a poly (ADP-ribose) polymerase (PARP) domain. duZAP was expressed in multiple duck tissues, with the highest mRNA expression in the spleen. Overexpression of duZAP in duck embryo fibroblast cells (DEFs) led to activation of the transcription factors IRF1 and NF-κB, and induction of IFN-ß. Analysis of deletion mutants revealed that both the WWE and PARP domains of duZAP were essential for activating the IFN-ß promoter. Knockdown of duZAP in DEFs significantly reduced poly (I:C)- and duck Tembusu virus (DTMUV)-induced IFN-ß activation. Our findings further the understanding of the role of duZAP in the duck innate immune response.


Assuntos
Proteínas Aviárias/metabolismo , Patos/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Células Cultivadas , Clonagem Molecular/métodos , Patos/genética , Patos/imunologia , Patos/virologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Imunidade Inata , Interferon beta/metabolismo , NF-kappa B/metabolismo , Filogenia , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Transdução de Sinais
11.
Front Immunol ; 12: 656700, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936086

RESUMO

SARS-CoV-2, the novel coronavirus infection has consistently shown an association with neurological anomalies in patients, in addition to its usual respiratory distress syndrome. Multi-organ dysfunctions including neurological sequelae during COVID-19 persist even after declining viral load. We propose that SARS-CoV-2 gene product, Spike, is able to modify the host exosomal cargo, which gets transported to distant uninfected tissues and organs and can initiate a catastrophic immune cascade within Central Nervous System (CNS). SARS-CoV-2 Spike transfected cells release a significant amount of exosomes loaded with microRNAs such as miR-148a and miR-590. microRNAs gets internalized by human microglia and suppress target gene expression of USP33 (Ubiquitin Specific peptidase 33) and downstream IRF9 levels. Cellular levels of USP33 regulate the turnover time of IRF9 via deubiquitylation. Our results also demonstrate that absorption of modified exosomes effectively regulate the major pro-inflammatory gene expression profile of TNFα, NF-κB and IFN-ß. These results uncover a bystander pathway of SARS-CoV-2 mediated CNS damage through hyperactivation of human microglia. Our results also attempt to explain the extra-pulmonary dysfunctions observed in COVID-19 cases when active replication of virus is not supported. Since Spike gene and mRNAs have been extensively picked up for vaccine development; the knowledge of host immune response against spike gene and protein holds a great significance. Our study therefore provides novel and relevant insights regarding the impact of Spike gene on shuttling of host microRNAs via exosomes to trigger the neuroinflammation.


Assuntos
COVID-19/metabolismo , Exossomos/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , MicroRNAs/metabolismo , Microglia/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Ubiquitina Tiolesterase/metabolismo , COVID-19/genética , COVID-19/fisiopatologia , COVID-19/virologia , Linhagem Celular , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/fisiopatologia , Sistema Nervoso Central/virologia , Endopeptidases/metabolismo , Exossomos/genética , Exossomos/patologia , Humanos , Inflamação/imunologia , Inflamação/virologia , Interferon beta/metabolismo , MicroRNAs/genética , Microglia/patologia , NF-kappa B/metabolismo , Estabilidade Proteica , Fator de Necrose Tumoral alfa/metabolismo
12.
Nat Commun ; 12(1): 2475, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931637

RESUMO

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNß. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.


Assuntos
Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , RNA Helicases DEAD-box/metabolismo , Infecções por HIV/imunologia , HIV-1/metabolismo , Imidazóis/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antirretrovirais/farmacologia , Apoptose/genética , Azepinas/química , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/química , Inibidores Enzimáticos/farmacologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Imidazóis/química , Hibridização in Situ Fluorescente , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Células Jurkat , Simulação de Acoplamento Molecular , RNA Viral/metabolismo , Análise de Célula Única , Survivina/metabolismo , Ativação Viral/efeitos dos fármacos , Replicação Viral/genética
13.
Arch Virol ; 166(8): 2173-2185, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34037855

RESUMO

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNß) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNß. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.


Assuntos
Imunidade Inata , Vírus da Bronquite Infecciosa/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Replicação Viral , Animais , Linhagem Celular , Galinhas , Chlorocebus aethiops , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Enzimas Desubiquitinantes/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Células Vero
14.
Front Immunol ; 12: 651254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897699

RESUMO

Interferon ß (IFN-ß) signaling activates the transcription factor complex ISGF3 to induce gene expression programs critical for antiviral defense and host immune responses. It has also been observed that IFN-ß activates a second transcription factor complex, γ-activated factor (GAF), but the significance of this coordinated activation is unclear. We report that in murine lung epithelial cells (MLE12) high doses of IFN-ß indeed activate both ISGF3 and GAF, which bind to distinct genomic locations defined by their respective DNA sequence motifs. In contrast, low doses of IFN-ß preferentially activate ISGF3 but not GAF. Surprisingly, in MLE12 cells GAF binding does not induce nearby gene expression even when strongly bound to the promoter. Yet expression of interferon stimulated genes is enhanced when GAF and ISGF3 are both active compared to ISGF3 alone. We propose that GAF may function as a dose-sensitive amplifier of ISG expression to enhance antiviral immunity and establish pro-inflammatory states.


Assuntos
Células Epiteliais/imunologia , Regulação da Expressão Gênica/imunologia , Fator Gênico 3 Estimulado por Interferon/metabolismo , Interferon beta/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Relação Dose-Resposta Imunológica , Células Epiteliais/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Multimerização Proteica/imunologia , RNA-Seq
15.
Front Immunol ; 12: 652965, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912177

RESUMO

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.


Assuntos
Imunidade Adaptativa/fisiologia , Linfócitos T CD4-Positivos/imunologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Interferon beta/metabolismo , Transdução de Sinais/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Adulto , Fatores Etários , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Humanos , Separação Imunomagnética , Recém-Nascido , Cultura Primária de Células , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Virology ; 559: 46-56, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33813212

RESUMO

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 Zinco
18.
Vet Microbiol ; 255: 109023, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33677368

RESUMO

The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays an important role in the innate immune response by the production of type I interferon (IFN) against DNA virus infection. However, viruses have evolved a variety of strategies to antagonize the host antiviral response to facilitate infection and replication. Pseudorabies virus (PRV), a DNA virus that causes great economic losses to the swine industry, encodes approximate 70 proteins, including some that are involved in evasion of host immunity. However, the mechanism employed by PRV to regulate type I IFN remains unclear. The results of the present study showed that the transcription levels of type I IFN were significantly upregulated by a UL24-deleted PRV strain. Furthermore, IFN-ß activation induced by poly(dA:dT) or stimulated by cGAS-STING was inhibited by UL24 overexpression in PK15 cells. Co-immunoprecipitation analysis demonstrated that UL24 interacts with and can degrade interferon regulatory factor 7 (IRF7) through the proteasome pathway in a dose-dependent manner. Together, these results showed that PRV UL24 interacted with IRF7 via the proteasome pathway and antagonized cGAS-STING-mediated activation of IFN-ß.


Assuntos
Herpesvirus Suídeo 1 , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , DNA Viral , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Fator Regulador 7 de Interferon/genética , Interferon beta/genética , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Plasmídeos , Suínos , Proteínas não Estruturais Virais
19.
mBio ; 12(2)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785626

RESUMO

Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.IMPORTANCE Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/ß receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs). In sum, these cellular functions establish a so-called antiviral state in infected and neighboring cells. To counteract these cellular defense mechanisms, viruses have evolved diverse strategies and encode gene products that target antiviral responses. Among such immune-evasive factors are viral microRNAs (miRNAs) that can interfere with host gene expression. We discovered that multiple miRNAs of Epstein-Barr virus (EBV) control over a dozen cellular genes that contribute to the antiviral states of immune cells, specifically B cells and plasmacytoid dendritic cells (pDCs). We identified the viral DNA genome as the activator of IFN-α and question the role of abundant EBV EBERs, that, contrary to previous reports, do not have an apparent inducing function in the IFN-I pathway early after infection.


Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Interferon-alfa/metabolismo , Interferon beta/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno , Humanos , Interferon-alfa/genética , Interferon beta/genética , MicroRNAs/genética , RNA Viral/genética , Transdução de Sinais , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo
20.
Biochem Biophys Res Commun ; 549: 207-213, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33684697

RESUMO

DNA transfection is routinely used for delivering expression of gene of interest to target cells. Transfected DNA has been known to activate cellular DNA sensor(s) and innate immune responses, but the effects of such responses on transfected DNA are not fully understood. STING (stimulator of interferon genes) is an important adaptor protein in cellular innate immune response to various DNA and RNA stimuli and upon activation induces significant type I interferon responses. In this work, we characterized the effects of STING on gene expression driven by transfected double-stranded DNA. We observed that gene expression from transfected DNA was repressed in the presence of overexpressed STING, but increased if endogenous STING was knocked down through RNA interference. Endogenous chromosomal genes and chromosome-integrated exogenous genes were not affected by such STING-mediated restriction, which did not depend on DNA circularity or linearity, promoter used, or bacterial sequences in transfected DNA. Mechanistically, STING-mediated repression of transfected DNA correlates with reduced mRNA levels, and partially involves the induction of interferon ß production by STING. Collectively, these data indicate that episomal double-stranded DNA is targeted by STING-mediated cell defense.


Assuntos
DNA/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Bases , Cromossomos Humanos/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interferon beta/metabolismo , Transfecção
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