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1.
Immunity ; 52(1): 109-122.e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882361

RESUMO

Recent work suggests that cholesterol metabolism impacts innate immune responses against infection. However, the key enzymes or the natural products and mechanisms involved are not well elucidated. Here, we have shown that upon DNA and RNA viral infection, macrophages reduced 7-dehydrocholesterol reductase (DHCR7) expression. DHCR7 deficiency or treatment with the natural product 7-dehydrocholesterol (7-DHC) could specifically promote phosphorylation of IRF3 (not TBK1) and enhance type I interferon (IFN-I) production in macrophages. We further elucidated that viral infection or 7-DHC treatment enhanced AKT3 expression and activation. AKT3 directly bound and phosphorylated IRF3 at Ser385, together with TBK1-induced phosphorylation of IRF3 Ser386, to achieve IRF3 dimerization. Deletion of DHCR7 and the DHCR7 inhibitors including AY9944 and the chemotherapy drug tamoxifen promoted clearance of Zika virus and multiple viruses in vitro or in vivo. Taken together, we propose that the DHCR7 inhibitors and 7-DHC are potential therapeutics against emerging or highly pathogenic viruses.


Assuntos
Desidrocolesteróis/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Macrófagos/imunologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estomatite Vesicular/imunologia , Células A549 , Animais , Linhagem Celular , Colesterol/metabolismo , Ativação Enzimática/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , Vírus da Estomatite Vesicular Indiana/imunologia
2.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 50(4): 540-545, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31642232

RESUMO

OBJECTIVE: To explore the anti-virus effect of AY358935 gene cloned by our research team on vesicular stomatitis virus (VSV), and studytheanti-virus mechanism. METHODS: HEK293 cells were stably transfected by the AY358935 gene recombinant plasmid pcDNA3.1-AY358935 or pcDNA3.1 blank plasmid respectively. Then VSV was added into the cell wells to infect the above cells at the multiplicity of infection (MOI) of 0.001. The virus titers in the liquid supernatant of the above three groups of cells were detected on different time, and the mortality of cells of each group was tested with trypan blue exclusion test at 24 h post VSV infection. Total RNA was extracted from the cells that stably transfected with target gene for the whole genome-wide cDNA microarray analysis. RESULTS: ① Virus titer:The virus titer in the liquid supernatant of pcDNA-3.1-AY358935 transfection cells group was obviously lower than those in pcDNA-3.1 transfection cell group and blank control cell group at 12 h post infection. The virus titerin the liquid supernatant of three groups were (7.16±2.33)×105 PFU/mL, (6.25±2.05)×106 PFU/mL and (7.75±2.54)×106 PFU/mL respectively at 18 h post infection. At that time, the virus titerin the liquid supernatant of pcDNA3.1-AY358935 group was nearly 10 times lower than those of other two groups (P < 0.01). ②Mortality of cells:The cell mortality of pcDNA3.1-AY358935 group, pcDNA3.1 group and blank group were (35.00±6.68)%, (78.33±15.03)% and (83.34±14.98)% respectively at 24 h post infection.The cell mortality of pcDNA3.1-AY358935 group was significantly decreased comparing with other two groups (P < 0.01). ③Result of genes chip analysis: compared with pcDNA3.1 group, 30 cell genes were up-regulated by more than 3 times in pcDNA3.1-AY358935 group. Among them, the proportion of interferon-activating gene, interferon-effect gene, cytokine and chemokine was 27%, 17%, and 20%, respectively. CONCLUSION: AY358935 gene hasan anti-VSV effect, and its anti-virus mechanism may involve the interferon-associated natural immune response.


Assuntos
Proteínas de Transporte/genética , Estomatite Vesicular/imunologia , Animais , Citocinas , Células HEK293 , Humanos , Interferons , Plasmídeos , Transfecção , Vesiculovirus
3.
PLoS Pathog ; 15(10): e1008093, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600344

RESUMO

ISG20 is a broad spectrum antiviral protein thought to directly degrade viral RNA. However, this mechanism of inhibition remains controversial. Using the Vesicular Stomatitis Virus (VSV) as a model RNA virus, we show here that ISG20 interferes with viral replication by decreasing protein synthesis in the absence of RNA degradation. Importantly, we demonstrate that ISG20 exerts a translational control over a large panel of non-self RNA substrates including those originating from transfected DNA, while sparing endogenous transcripts. This activity correlates with the protein's ability to localize in cytoplasmic processing bodies. Finally, these functions are conserved in the ISG20 murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. Overall, our results posit ISG20 as an important defense factor able to discriminate the self/non-self origins of the RNA through translation modulation.


Assuntos
Antivirais/farmacologia , Exorribonucleases/farmacologia , Biossíntese de Proteínas , RNA Viral/metabolismo , Estomatite Vesicular/imunologia , Vesiculovirus/imunologia , Replicação Viral/efeitos dos fármacos , Animais , Exorribonucleases/fisiologia , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Estabilidade de RNA , RNA Viral/genética , Estomatite Vesicular/tratamento farmacológico , Estomatite Vesicular/virologia , Vesiculovirus/efeitos dos fármacos
4.
Biochem Biophys Res Commun ; 511(2): 287-293, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30795865

RESUMO

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-ß mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-ß and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Imunidade Inata , Interferon beta/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Apoptose , Linhagem Celular , Humanos , Membranas Mitocondriais/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
5.
Mol Cell ; 73(4): 803-814.e6, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639243

RESUMO

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.


Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Íntrons , Macrófagos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estomatite Vesicular/metabolismo , Vírus da Estomatite Vesicular Indiana/patogenicidade , Animais , Sítios de Ligação , Sequência Rica em GC , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Ligação Proteica , Sítios de Splice de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Células Vero , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia
6.
Can J Vet Res ; 82(4): 316-321, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30363380

RESUMO

Foot-and-mouth disease (FMD) and vesicular stomatitis (VS) cause such similar clinical signs and lesions that laboratory tests are required to distinguish between infections caused by each virus. Using mouse anti-foot-and-mouth disease virus (FMDV) 3B monoclonal or polyclonal anti-vesicular stomatitis virus-New Jersey (VSV-NJ) antibodies and recombinant FMDV 3ABC or VSV-NJ glycoprotein (G) antigens coated to MagPlex beads, competitive Luminex immunoassays (cLIAs) were developed for FMDV and VSV-NJ, respectively. The cLIAs successfully detected antibodies to FMDV 3ABC and VSV-NJ G in sera from infected animals. The diagnostic sensitivity and specificity were 93% and 98%, respectively for FMDV and 93% and 95.4%, respectively for VSV-NJ. These cLIAs are potential alternatives for competitive enzyme-linked immunosorbent assays (cELISAs) and provide the opportunity for multiplexing to reduce time and the amount of serum required for testing.


Assuntos
Anticorpos Antivirais/sangue , Febre Aftosa/diagnóstico , Imunoensaio/veterinária , Estomatite Vesicular/diagnóstico , Animais , Diagnóstico Diferencial , Febre Aftosa/imunologia , Vírus da Febre Aftosa/imunologia , Sensibilidade e Especificidade , Especificidade da Espécie , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
7.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30232190

RESUMO

Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example, Cocal virus, have been proposed as alternative LV envelopes with possible advantages over VSVind.G. Well-characterized antibodies that recognize VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here, we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralizes G proteins from three strains of VSV, as well as Cocal and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralizes only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Reações Cruzadas , Epitopos/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Testes de Neutralização , Filogenia , Homologia de Sequência , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
8.
Microbiol Immunol ; 62(9): 585-593, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30160073

RESUMO

MicroRNAs are short, non-coding RNAs that have been shown to regulate a wide range of biological processes, including host antiviral immune responses. In the present study, microRNA-92a (miR-92a) was identified as a negative regulator in macrophage-mediated antiviral responses. Overexpression of miR-92a decreases vesicular stomatitis virus (VSV)-induced production of type-I IFNs and facilitates viral replication in macrophages. The mechanism is that miR-92a directly targets RIG-I and reduces its expression, thereby attenuating VSV-triggered activation of TBK-binding kinase 1 and IRF3, both of which are crucial for initiating transcription of type-I IFN genes. Our results demonstrate for the first time the novel role of miR-92a in suppressing antiviral innate immunity.


Assuntos
Proteína DEAD-box 58/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Antivirais/metabolismo , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7/efeitos dos fármacos , Alinhamento de Sequência , Regulação para Cima/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
9.
Biochem Biophys Res Commun ; 498(4): 891-897, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29545178

RESUMO

Type-I interferons (IFN-I) are widely used for antiviral immunotherapy in clinic. Therefore, identification of the regulators of IFN-I antiviral activity is important for developing novel targets for IFN-based antiviral therapy. Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1) is critical for cellular inflammatory responses. However, the roles of MCPIP1 in interferons (IFNs)-mediated antiviral immunity are unexplored. In this study, we demonstrate for the first time that MCPIP1 is an important positive regulator of IFNs antiviral activity. We found that MCPIP1 can promote innate antiviral immunity independently of both its RNase and deubiquitinase activity. Furthermore, we reveal that MCPIP1 is an IFN-induced positive feedback signal molecule which promotes IFN-I-mediated antiviral efficacy. Mechanistically, MCPIP1 does not affect the activation of JAK/STAT upstream of IFN-I signaling, but significantly promotes IFN-I signaling by enhancing ISRE promoter activity and expression of interferon-stimulated genes (ISGs). And MCPIP1-mediated activation of IFN-I signaling is independently of its RNase and deubiquitinase activity. These findings uncover a novel innate antiviral mechanism mediated by the IFN-MCPIP1 axis, and may provide potential targets for enhancing IFNs antiviral therapy.


Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Ribonucleases/imunologia , Fatores de Transcrição/imunologia , Viroses/imunologia , Linhagem Celular , Humanos , Interferon Tipo I/genética , Regiões Promotoras Genéticas , Elementos de Resposta/genética , Transdução de Sinais/imunologia , Ativação Transcricional , Transfecção , Estomatite Vesicular/imunologia , Vesiculovirus/imunologia
10.
J Vet Diagn Invest ; 30(4): 510-516, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29595090

RESUMO

Vesicular stomatitis (VS) is a vesicular disease of horses, cattle, and pigs in the Western Hemisphere caused by viruses in the genus Vesiculovirus. Disease manifests as vesicles and erosions on the oral mucosa, teats, prepuce, and coronary band, and is similar in presentation to foot-and-mouth disease. Laboratory confirmation is therefore required. Conventional assays include competitive (c)ELISA and complement fixation (CF). The cELISA provides more accurate herd-level detection of VSV-exposed cattle, but may lack the ability to capture fluctuating antibody levels in individual animals. The CF assay can confirm newly infected animals because of its ability to detect antigen-antibody complexes, thus is considered to be indicative of IgM. We evaluated the immune status of 2 herds affected by VSV in 2014 by testing sera collected in June 2015. Two conventional assays were compared to a novel IgM-IgG ELISA. When sampled in 2015, both herds had detectable VSV-specific antibodies; 18% and 36% of animals tested by cELISA and 2% and 8% of animals tested by CF were positive. The novel IgM-IgG assay exhibited fair agreement (adjusted kappa score of 48) with the conventional assays, and should be evaluated further to assess its ability to replace the 2 separate assays with a single assay system, or for its ability to replace the CF assay as a more sensitive method for defining newly exposed animals.


Assuntos
Doenças dos Bovinos/epidemiologia , Surtos de Doenças/veterinária , Estomatite Vesicular/epidemiologia , Vesiculovirus/imunologia , Animais , Anticorpos Antivirais/sangue , Bioensaio/veterinária , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/imunologia , Colorado/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Masculino , Estudos Soroepidemiológicos , Estomatite Vesicular/sangue , Estomatite Vesicular/diagnóstico , Estomatite Vesicular/imunologia
11.
Hum Vaccin Immunother ; 14(4): 994-1002, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29206076

RESUMO

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.


Assuntos
Artrópodes/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Mosquitos Vetores/imunologia , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Aedes/imunologia , Aedes/virologia , Animais , Artrópodes/virologia , Culex/imunologia , Culex/virologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vero , Estomatite Vesicular/imunologia , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/virologia
12.
J Virol ; 92(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29142134

RESUMO

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.


Assuntos
Interferon Tipo I/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Estomatite Vesicular/imunologia , Imunidade Adaptativa , Animais , Imunidade Inata , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Fator de Transcrição RelA/metabolismo , Vesiculovirus/fisiologia , Replicação Viral
13.
Nat Immunol ; 18(10): 1094-1103, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28846086

RESUMO

DEAD-box (DDX) helicases are vital for the recognition of RNA and metabolism and are critical for the initiation of antiviral innate immunity. Modification of RNA is involved in many biological processes; however, its role in antiviral innate immunity has remained unclear. Here we found that nuclear DDX member DDX46 inhibited the production of type I interferons after viral infection. DDX46 bound Mavs, Traf3 and Traf6 transcripts (which encode signaling molecules involved in antiviral responses) via their conserved CCGGUU element. After viral infection, DDX46 recruited ALKBH5, an 'eraser' of the RNA modification N6-methyladenosine (m6A), via DDX46's DEAD helicase domain to demethylate those m6A-modified antiviral transcripts. It consequently enforced their retention in the nucleus and therefore prevented their translation and inhibited interferon production. DDX46 also suppressed antiviral innate immunity in vivo. Thus, DDX46 inhibits antiviral innate responses by entrapping selected antiviral transcripts in the nucleus by erasing their m6A modification, a modification normally required for export from the nucleus and translation.


Assuntos
Adenina/análogos & derivados , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Imunidade Inata/genética , Transcrição Genética , Adenina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interferon Tipo I/biossíntese , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Motivos de Nucleotídeos , Ligação Proteica , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Replicação Viral
14.
J Immunol ; 199(4): 1372-1381, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28687662

RESUMO

Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel-dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-ß expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-ß secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Interferon beta/biossíntese , Receptores Purinérgicos P2X7/metabolismo , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Vírus da Leucemia Murina/efeitos dos fármacos , Vírus da Leucemia Murina/imunologia , Medições Luminescentes , Camundongos , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/imunologia , Células RAW 264.7 , Receptores Purinérgicos P2X7/imunologia , Transdução de Sinais , Simplexvirus/efeitos dos fármacos , Simplexvirus/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/efeitos dos fármacos
15.
J Infect Dis ; 215(12): 1789-1798, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28549145

RESUMO

Background: This study (NCT02503202) evaluated the safety of recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein vaccine (rVSVΔG-ZEBOV-GP). Methods: Overall, 1197 subjects were randomized 2:2:2:2:1; 1194 were vaccinated with 1 dose of 1 of 3 lots of rVSVΔG- ZEBOV-GP (2 × 107 plaque-forming units [pfu], n = 797; combined-lots group), a single high-dose lot of rVSVΔG-ZEBOV-GP (1 × 108 pfu, n = 264; high-dose group), or placebo (n = 133). Daily temperatures and adverse events (AEs) were recorded days 1 to 42 postvaccination. Solicited AEs included injection-site AEs from days 1 to 5, and joint pain, joint swelling, vesicular lesions (blisters), and rashes from days 1 to 42. Serious AEs (SAEs) were recorded through 6 months postvaccination. Results: Fever (≥38.0°C) was observed in 20.2% of combined lots (3.2% with ≥39.0°C), 32.2% of high-dose (4.3% with ≥39.0°C), and 0.8% of placebo (0.8% with ≥39.0°C). Incidences of AEs of interest (days 1-42) were arthralgia (17.1% combined lots, 20.4% high-dose, 3.0% placebo), arthritis (5.1% combined lots, 4.2% high-dose, 0.0% placebo), and rash (3.8% combined lots, 3.8% high-dose, 1.5% placebo). Twenty-one SAEs and 2 deaths were reported, all assessed by investigators as unrelated to vaccine. Conclusions: rVSVΔG-ZEBOV-GP was generally well-tolerated, with increased rates of injection-site and systemic AEs compared to placebo, and no vaccine-related SAEs or deaths. These findings support the use of rVSVΔG-ZEBOV-GP vaccine in persons at risk for Ebola virus disease. Clinical Trials Registration: NCT02503202.


Assuntos
Vacinas contra Ebola/efeitos adversos , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Estomatite Vesicular/imunologia , Proteínas do Envelope Viral/imunologia , Adolescente , Adulto , Idoso , Método Duplo-Cego , Vacinas contra Ebola/imunologia , Feminino , Voluntários Saudáveis/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Vacinação/métodos , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/efeitos adversos , Adulto Jovem
16.
Mol Ther ; 25(8): 1900-1916, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28527723

RESUMO

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.


Assuntos
Autofagia , Fator 2 Relacionado a NF-E2/metabolismo , Vírus Oncolíticos/fisiologia , Transdução de Sinais , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Terapia Combinada , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Isotiocianatos/farmacologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Neoplasias/metabolismo , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas da Matriz Viral/genética , Replicação Viral/efeitos dos fármacos
17.
Immunology ; 152(1): 102-114, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28464285

RESUMO

As the most important host defence against viral infection, interferon (IFN) stimulates hundreds of antiviral genes (ISGs) that together establish an 'antiviral state'. However, the antiviral function of most ISGs in viral infection still need further exploration. Here, we demonstrated that the expression of G-protein-coupled receptor 146 (GPR146) is highly increased by both IFN-ß and IFN-γ in a signal transducer and activator of transcription 1-dependent signalling pathway. Most importantly, overexpression of GPR146 protects the host cells from vesicular stomatitis virus and Newcastle disease virus infection but not from infection by herpes simplex virus. In contrast, the virus-induced IFN-ß production changed little in Gpr146-knockout cells. Furthermore, the Gpr146-deficient mice showed similar susceptibility to wild-type mice with vesicular stomatitis virus infection. Interestingly, the expression of GPR146 in virus-infected cells was strikingly reduced and can partially explain why the viral infection was little influenced in Gpr146-knockout mice. Surprisingly, virus-activated IFN regulatory factor 3 (IRF3) signalling not only induces the expression of IFN but also represses GPR146 expression through HES1 (hairy and enhancer of split-1)-mediated transcriptional activity to establish a dynamic equilibrium between pro-viral and antiviral stages in host cells. Taken together, these data reveal the antiviral role of GPR146 in fighting viral infection although the GPR146-mediated protection is eliminated by IRF3/HES1-signalling, which suggests a potential therapeutic significance of both GPR146 and HES1 signalling in viral infection.


Assuntos
Herpes Simples/prevenção & controle , Fator Regulador 3 de Interferon/metabolismo , Macrófagos Peritoneais/metabolismo , Doença de Newcastle/prevenção & controle , Receptores Acoplados a Proteínas-G/deficiência , Transdução de Sinais , Fatores de Transcrição HES-1/metabolismo , Estomatite Vesicular/prevenção & controle , Animais , Genótipo , Células HEK293 , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/imunologia , Interferon beta/farmacologia , Interferon gama/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Newcastle/imunologia , Doença de Newcastle/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/metabolismo , Fenótipo , Células RAW 264.7 , Interferência de RNA , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Fatores de Transcrição HES-1/imunologia , Transfecção , Células Vero , Estomatite Vesicular/imunologia , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação Viral
18.
Vaccine ; 35(41): 5481-5486, 2017 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-28427845

RESUMO

Development of vaccines against highly pathogenic viruses that could also be used as agents of bioterrorism is both a public health issue and a national security priority. Methods that can quantify neutralizing antibodies will likely be crucial in demonstrating vaccine effectiveness, as most licensed viral vaccines are effective due to their capacity to elicit neutralizing antibodies. Assays to determine whether antibodies are neutralizing traditionally involve infectious virus, and the assay most commonly used is the plaque-reduction neutralization test (PRNT). However, when the virus is highly pathogenic, this assay must be done under the appropriate level of containment; for tier one select agents, such as Ebola virus (EBOV), it is performed under Biological Safety Level 4 (BSL-4) conditions. Developing high-throughput neutralization assays for these viruses that can be done in standard BSL-2 laboratories should facilitate vaccine development. Our approach is to use a replication-competent hybrid virus whose genome carries the envelope gene from the pathogenic virus on the genetic backbone of a non-pathogenic virus, such as vesicular stomatitis virus (VSV). We have generated hybrid VSVs carrying the envelope genes for several species of ebolavirus. The readout for infectivity is a one-step reverse transcriptase quantitative PCR (RT-qPCR), an approach that we have used for other viruses that allows robustness and adaptability to automation. Using this method, we have shown that neutralization can be assessed within 6-16h after infection. Importantly, the titers obtained in our assay with two characterized antibodies were in agreement with titers obtained in other assays. Finally, although in this paper we describe the VSV platform to quantify neutralizing antibodies to ebolaviruses, the platform should be directly applicable to any virus whose envelope is compatible with VSV biology.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Células HEK293 , Humanos , Testes de Neutralização/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Células Vero , Estomatite Vesicular/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia
19.
J Exp Med ; 214(5): 1547-1555, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28420733

RESUMO

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Interferon-alfa/sangue , Humanos , Fatores Reguladores de Interferon/sangue , Fatores Reguladores de Interferon/líquido cefalorraquidiano , Interferon-alfa/líquido cefalorraquidiano , Lúpus Eritematoso Sistêmico/sangue , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Linfócitos T/metabolismo , Estomatite Vesicular/imunologia
20.
J Biol Chem ; 292(1): 292-304, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-27879319

RESUMO

The host protein Stimulator of Interferon Genes (STING) has been shown to be essential for recognition of both viral and intracellular bacterial pathogens, but its regulation remains unclear. Previously, we reported that mitochondrial membrane potential regulates STING-dependent IFN-ß induction independently of ATP synthesis. Because mitochondrial membrane potential controls calcium homeostasis, and AMP-activated protein kinase (AMPK) is regulated, in part, by intracellular calcium, we postulated that AMPK participates in STING activation; however, its role has yet to be been defined. Addition of an intracellular calcium chelator or an AMPK inhibitor to either mouse macrophages or mouse embryonic fibroblasts (MEFs) suppressed IFN-ß and TNF-α induction following stimulation with the STING-dependent ligand 5,6-dimethyl xanthnone-4-acetic acid (DMXAA). These pharmacological findings were corroborated by showing that MEFs lacking AMPK activity also failed to up-regulate IFN-ß and TNF-α after treatment with DMXAA or the natural STING ligand cyclic GMP-AMP (cGAMP). As a result, AMPK-deficient MEFs exhibit impaired control of vesicular stomatitis virus (VSV), a virus sensed by STING that can cause an influenza-like illness in humans. This impairment could be overcome by pretreatment of AMPK-deficient MEFs with type I IFN, illustrating that de novo production of IFN-ß in response to VSV plays a key role in antiviral defense during infection. Loss of AMPK also led to dephosphorylation at Ser-555 of the known STING regulator, UNC-51-like kinase 1 (ULK1). However, ULK1-deficient cells responded normally to DMXAA, indicating that AMPK promotes STING-dependent signaling independent of ULK1 in mouse cells.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Antivirais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Imunidade Inata/imunologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/virologia , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Macrófagos Peritoneais , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Estomatite Vesicular/imunologia , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia
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