RESUMO
The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , TYK2 Quinase , Carrapatos , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Interferons/metabolismo , Carrapatos/metabolismo , TYK2 Quinase/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , HumanosRESUMO
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.
Assuntos
Processamento Alternativo , Dependovirus/genética , Distrofina/genética , Distrofia Muscular Animal/terapia , Oligorribonucleotídeos Antissenso/genética , RNA Nuclear Pequeno/genética , Animais , Sequência de Bases , Cálcio/metabolismo , Cães , Éxons , Membro Anterior/fisiopatologia , Terapia Genética , Vetores Genéticos/administração & dosagem , Injeções Intramusculares , Dados de Sequência Molecular , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Força Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Transcrição Gênica , Utrofina/genética , Utrofina/metabolismoRESUMO
Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 µg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.
Assuntos
Bradicinina/fisiologia , Endotélio Vascular/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Pressão Sanguínea , Artérias Carótidas/enzimologia , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Cães , Distrofia Muscular de Duchenne/enzimologia , Óxido Nítrico/fisiologia , Disfunção Ventricular EsquerdaRESUMO
BACKGROUND: Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus. Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells. METHODS: To investigate virus-vector protein-protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV. RESULTS: For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways. CONCLUSION: We provide the first description of the TBEV/LIV-I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses.
Assuntos
Proteínas de Artrópodes/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Interações entre Hospedeiro e Microrganismos , Ixodes/genética , Ixodes/virologia , Proteínas Virais/metabolismo , Animais , Proteínas de Artrópodes/genética , Feminino , Biblioteca Gênica , Fases de Leitura Aberta , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/genéticaRESUMO
To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.
Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Transgenes/genética , Transgenes/imunologia , Administração Oral , Animais , Feminino , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Especificidade de Órgãos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fagócitos/metabolismo , Transporte Proteico , VacinaçãoRESUMO
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC), is a devastating respiratory disease of cattle. In sub-Saharan Africa, where CBPP is enzootic, live attenuated vaccines are deployed but afford only short-lived protection. In cattle, recovery from experimental MmmSC infection has been associated with the presence of CD4(+) T lymphocytes that secrete interferon gamma in response to MmmSC, and in particular to the lipoprotein A (LppA) antigen. In an effort to develop a better vaccine against CBPP, a viral vector (Ad5-LppA) that expressed LppA was generated from human adenovirus type 5. The LppA-specific immune responses elicited by the Ad5-LppA vector were evaluated in mice, and compared to those elicited by recombinant LppA formulated with a potent adjuvant. Notably, a single administration of Ad5-LppA, but not recombinant protein, sufficed to elicit a robust LppA-specific humoral response. After a booster administration, both vector and recombinant protein elicited strong LppA-specific humoral and cell-mediated responses. Ex vivo stimulation of splenocytes induced extensive proliferation of CD4(+) T cells for mice immunized with vector or protein, and secretion of T helper 1-associated and proinflammatory cytokines for mice immunized with Ad5-LppA. Our study - by demonstrating the potential of a viral-vectored prototypic vaccine to elicit prompt and robust immune responses against a major antigen of MmmSC - represents a first step in developing a recombinant vaccine against CBPP.
Assuntos
Adenovírus Humanos/genética , Vacinas Bacterianas/imunologia , Portadores de Fármacos , Vetores Genéticos , Lipoproteína(a)/imunologia , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Citocinas/metabolismo , Feminino , Humanos , Lipoproteína(a)/biossíntese , Lipoproteína(a)/genética , Camundongos Endogâmicos BALB C , Mycoplasma mycoides/genéticaRESUMO
The GRMD (Golden retriever muscular dystrophy) dog has been widely used in pre-clinical trials targeting DMD (Duchenne muscular dystrophy), using in many cases a concurrent immune-suppressive treatment. The aim of this study is to assess if such a treatment could have an effect on the disease course of these animals. Seven GRMD dogs were treated with an association of cyclosporine A (immunosuppressive dosage) and prednisolone (2 mg/kg/d) during 7 months, from 2 to 9 months of age. A multi-parametric evaluation was performed during this period which allowed us to demonstrate that this treatment had several significant effects on the disease progression. The gait quality as assessed by 3D-accelerometry was dramatically improved. This was consistent with the evolution of other parameters towards a significant improvement, such as the clinical motor score, the post-tetanic relaxation and the serum CK levels. In contrast the isometric force measurement as well as the histological evaluation argued in favor of a more severe disease progression. In view of the disease modifying effects which have been observed in this study it should be concluded that immunosuppressive treatments should be used with caution when carrying out pre-clinical studies in this canine model of DMD. They also highlight the importance of using a large range of multi-parametric evaluation tools to reliably draw any conclusion from trials involving dystrophin-deficient dogs, which reproduce the complexity of the human disease.
Assuntos
Imunossupressores/uso terapêutico , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Acelerometria , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Creatina Quinase/sangue , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Cães , Seguimentos , Marcha/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Atividade Motora/efeitos dos fármacos , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/complicações , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/fisiopatologia , Análise de Componente Principal , Tetania/sangue , Tetania/complicações , Tetania/fisiopatologiaRESUMO
AIMS: Cardiomyopathy is a lethal result of Duchenne muscular dystrophy (DMD), but its characteristics remain elusive. The golden retriever muscular dystrophy (GRMD) dogs produce DMD pathology and mirror DMD patient's symptoms, including cardiomyopathy. We previously showed that bradykinin slows the development of pacing-induced heart failure. Therefore, the goals of this research were to characterize dystrophin-deficiency cardiomyopathy and to examine cardiac effects of bradykinin in GRMD dogs. METHODS AND RESULTS: At baseline, adult GRMD dogs had reduced fractional shortening (28 ± 2 vs. 38 ± 2% in control dogs, P < 0.001) and left ventricular (LV) subendocardial dysfunction leading to impaired endo-epicardial gradient of radial systolic velocity (1.3 ± 0.1 vs. 3.8 ± 0.2 cm/s in control dogs, P < 0.001) measured by echocardiography. These changes were normalized by bradykinin infusion (1 µg/min, 4 weeks). In isolated permeabilized LV subendocardial cells of GRMD dogs, tension-calcium relationships were shifted downward and force-generating capacity and transmural gradient of myofilament length-dependent activation were impaired compared with control dogs. Concomitantly, phosphorylation of sarcomeric regulatory proteins and levels of endothelial and neuronal nitric oxide synthase (e/nNOS) in LV myocardium were significantly altered in GRMD dogs. All these abnormalities were normalized in bradykinin-treated GRMD dogs. CONCLUSIONS: Cardiomyopathy in GRMD dogs is characterized by profound LV subendocardial dysfunction, abnormal sarcomeric protein phosphorylation, and impaired e/nNOS, which can be normalized by bradykinin treatment. These data provide new insights into the pathophysiological mechanisms accounting for DMD cardiomyopathy and open new therapeutic perspectives.