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1.
PLoS Pathog ; 11(12): e1005350, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26717518

RESUMO

Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I-like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.


Assuntos
Vírus da Dengue/metabolismo , Dengue/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Interações Hospedeiro-Parasita/imunologia , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/imunologia
2.
J Infect Dis ; 212(12): 2011-20, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26063222

RESUMO

Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P < .001). This effect was due to DENV-2 infections (OR, 0.13; P = .007) but was absent for DENV-1, in accordance with the serotype-dependent OAS3 activity found in the functional study. Severe dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , Vírus da Dengue/imunologia , Dengue/patologia , Predisposição Genética para Doença , 2',5'-Oligoadenilato Sintetase/metabolismo , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Dengue/genética , Dengue/imunologia , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Adulto Jovem
3.
PLoS Pathog ; 11(3): e1004750, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25816318

RESUMO

Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. ß-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired ß-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid ß-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA ß-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and ß subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA ß-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA ß-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA ß-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA ß-oxidation and inducing cytokine expression by association with MTP.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/metabolismo , Ácidos Graxos/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/genética , Ácidos Graxos/genética , Células HEK293 , Humanos , Camundongos , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Oxirredução , Mutação Puntual , Proteínas não Estruturais Virais/genética
4.
Nucleic Acids Res ; 41(5): 3314-26, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23355615

RESUMO

Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), belonging to the MCPIP family with highly conserved CCCH-type zinc finger and Nedd4-BP1, YacP Nuclease domains, has been implicated in negative regulation of the cellular inflammatory responses. In this report, we demonstrate for the first time that this RNA-binding nuclease also targets viral RNA and possesses potent antiviral activities. Overexpression of the human MCPIP1, but not MCPIP2, MCPIP3 or MCPIP4, inhibited Japanese encephalitis virus (JEV) and dengue virus (DEN) replication. The functional analysis of MCPIP1 revealed that the activities of RNase, RNA binding and oligomerization, but not deubiqutinase, are required for its antiviral potential. Furthermore, infection of other positive-sense RNA viruses, such as sindbis virus and encephalomyocarditis virus, and negative-sense RNA virus, such as influenza virus, as well as DNA virus, such as adenovirus, can also be blocked by MCPIP1. Moreover, the endogenous MCPIP1 gene expression was induced by JEV and DEN infection, and knockdown of MCPIP1 expression enhanced the replication of JEV and DEN in human cells. Thus, MCPIP1 can act as a host innate defense via RNase activity for targeting and degrading viral RNA.


Assuntos
Vírus da Dengue/fisiologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Estabilidade de RNA , RNA Viral/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/imunologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Ribonucleases , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Replicação Viral , Dedos de Zinco
5.
J Immunol ; 183(12): 8035-43, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19923450

RESUMO

The 2',5'-oligoadenylate synthetase (OAS) and its downstream effector RNase L play important roles in host defense against virus infection. Oas1b, one of the eight Oas1 genes in the mouse genome, has been identified as a murine flavivirus-resistance gene. Four genes, OAS1, OAS2, OAS3, and OAS-like (OASL), have been identified in the human OAS gene family, and 10 isoforms, including OAS1 (p42, p44, p46, p48, and p52), OAS2 (p69 and p71), OAS3 (p100), and OASL (p30 and p59) can be generated by alternative splicing. In this study, we determined the role of the human OAS/RNase L pathway in host defense against dengue virus (DEN) infection and assessed the antiviral potential of each isoform in the human OAS family. DEN replication was reduced by overexpression and enhanced by knockdown of RNase L expression, indicating a protective role for RNase L against DEN replication in human cells. The human OAS1 p42, OAS1 p46, and OAS3 p100, but not the other OAS isoforms, blocked DEN replication via an RNase L-dependent mechanism. Furthermore, the anti-DEN activities of these three OAS isoforms correlated with their ability to trigger RNase L activation in DEN-infected cells. Thus, OAS1 p42/p46 and OAS3 p100 are likely to contribute to host defense against DEN infection and play a role in determining the outcomes of DEN disease severity.


Assuntos
2',5'-Oligoadenilato Sintetase/fisiologia , Vírus da Dengue/imunologia , Dengue/enzimologia , Dengue/prevenção & controle , Família Multigênica , 2',5'-Oligoadenilato Sintetase/biossíntese , 2',5'-Oligoadenilato Sintetase/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Dengue/imunologia , Dengue/virologia , Endorribonucleases/genética , Endorribonucleases/metabolismo , Endorribonucleases/fisiologia , Ativação Enzimática/imunologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Ativação Viral/imunologia
6.
J Virol ; 80(12): 5908-18, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16731929

RESUMO

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes severe human disease, has been shown to block the interferon (IFN)-induced Janus kinase signal transducer and activation of transcription (Jak-Stat) signaling cascade by preventing Tyk2 tyrosine phosphorylation and Stat activation. In this study, we demonstrate that expression of the JEV nonstructural protein NS5 readily blocked IFN-stimulated Jak-Stat signaling events such as Stat1 nuclear translocation and tyrosine phosphorylation of Tyk2 and Stat1. The region of JEV NS5 responsible for Stat1 suppression was identified using various deletion clones. Deletion of 83 N-terminal residues of JEV NS5, but not the 143 C-terminal residues, abolished its ability to block IFN-stimulated Stat1 activation. The role of JEV NS5 as an IFN antagonist was further demonstrated by its ability to block the induction of interferon-stimulated genes and the antiviral effect of IFN-alpha against the IFN-sensitive encephalomyocarditis virus, which appears to replicate and kill cells that express NS5 even with alpha IFN treatment. Furthermore, the molecular mechanism responsible for IFN antagonism by NS5 probably involves protein tyrosine phosphatases (PTPs), as the IFN-blocking events in both JEV-infected and NS5-expressing cells were reversed by sodium orthovanadate, a broad-spectrum inhibitor of PTPs. We suggest that JEV NS5 is an IFN antagonist and that it may play a role in blocking IFN-stimulated Jak-Stat signaling via activation of PTPs during JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Interferons/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação da Expressão Gênica , Humanos , Interferons/genética , Interferons/farmacologia , Janus Quinase 1 , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Células Vero
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