Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Viruses ; 14(11)2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36423130

RESUMO

Hepatitis C virus (HCV) core protein is a multifunctional protein that is involved in the proliferation, inflammation, and apoptosis mechanism of hepatocytes. HCV core protein genetic variability has been implicated in various outcomes of HCV pathology and treatment. In the present study, we aimed to analyze the role of the HCV core protein in tumor necrosis factor α (TNFα)-induced death under the viewpoint of HCV genetic variability. Immortalized hepatocytes (IHH), and not the Huh 7.5 hepatoma cell line, stably expressing HCV subtype 4a and HCV subtype 4f core proteins showed that only the HCV 4a core protein could increase sensitivity to TNFα-induced death. Development of two transgenic mice expressing the two different core proteins under the liver-specific promoter of transthyretin (TTR) allowed for the in vivo assessment of the role of the core in TNFα-induced death. Using the TNFα-dependent model of lipopolysaccharide/D-galactosamine (LPS/Dgal), we were able to recapitulate the in vitro results in IHH cells in vivo. Transgenic mice expressing the HCV 4a core protein were more susceptible to the LPS/Dgal model, while mice expressing the HCV 4f core protein had the same susceptibility as their littermate controls. Transcriptome analysis in liver biopsies from these transgenic mice gave insights into HCV core molecular pathogenesis while linking HCV core protein genetic variability to differential pathology in vivo.


Assuntos
Hepacivirus , Hepatite C , Camundongos , Animais , Hepacivirus/genética , Fator de Necrose Tumoral alfa/metabolismo , Lipopolissacarídeos/metabolismo , Hepatite C/metabolismo , Hepatócitos , Genótipo , Camundongos Transgênicos
2.
Viruses ; 14(8)2022 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-36016316

RESUMO

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein-known as ARFP (alternative reading frame protein) or Core+1 protein-is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.


Assuntos
Carcinoma Hepatocelular , Hepatite C , Hepacivirus/genética , Hepacivirus/metabolismo , Antígenos da Hepatite C , Humanos , Isoformas de Proteínas/metabolismo , Proteômica , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
3.
Germs ; 12(3): 384-393, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37680672

RESUMO

Introduction: Hepatitis C virus (HCV) infection is a prime cause of chronic hepatitis worldwide, that often silently progresses to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Notably, the majority of individuals infected with HCV develop symptoms at late stages, often associated with liver damage that cannot revert after virus clearance. Thus, current antiviral therapy alone is rather insufficient to eliminate the global burden of HCV in the near future.During the past few years, vitamin D deficiency as well as certain single nucleotide polymorphisms in the vitamin D receptor (VDR) gene have been associated with liver fibrosis. Therefore, the aim of the present study was to investigate the possible correlation between VDR polymorphisms ApaI (rs7975232) and TaqI (rs731236) and the fibrosis stage of patients with HCV infection from Thrace, Greece. Methods: Eighty-one patients with HCV infection underwent transient elastography for the assessment of their fibrosis stage, and PCR-restriction fragment length polymorphism (RFLP) genotyping for VDR ApaI and TaqI polymorphisms. VDR genotypes were then statistically associated with the patients' fibrosis stage using ordinal regression models. Results: Non-cirrhotic stages were positively correlated with TaqI TT genotype (p=0.003) and negatively correlated with TaqI TC genotype (p=0.007). In the presence of Hardy-Weinberg equilibrium and linkage disequilibrium between the two VDR polymorphisms, mild fibrosis stages (F0-2) were correlated with ApaI/TaqI GG/TT (p=0.002) and TG/TT (p=0.008) genotypes, while cirrhotic stage F4 was associated with ApaI/TaqI TG/TC genotype (p=0.038). Conclusions: TaqI TT and ApaI/TaqI GG/TT, TG/TT and TG/TC genotypes could be explored as prognostic genetic markers for fibrosis susceptibility in HCV patients.

4.
Int J Mol Sci ; 21(18)2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32972019

RESUMO

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5'-untranslated region (5'UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597-599 and 603-605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344-596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome-HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5'UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.


Assuntos
Hepacivirus/metabolismo , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , RNA Viral/metabolismo , Proteínas do Core Viral/biossíntese , Linhagem Celular Tumoral , Códon de Iniciação , Hepacivirus/genética , Humanos , RNA Viral/genética , Proteínas do Core Viral/genética
5.
Ann Gastroenterol ; 32(1): 88-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30598597

RESUMO

BACKGROUND: Intergenotypic recombinant hepatitis C virus (HCV) strains emerge rarely during coinfection of the same individual with two HCV genotypes. Few recombinant HCV strains have been identified to date and only one, CRF01 2k/1b, has become a worldwide concern. This study reevaluated the genotyping of three HCV genotype 2 strains from a group of patients with an unusually low rate of sustained virological response after pegylated interferon/ribavirin treatment. In addition, genetic determinants of host interferon resistance were evaluated. METHODS: The HCV type 2 strains from the patients' serum were subjected to partial sequencing of the core-E1, NS2, NS5A and NS5B regions by reverse transcription polymerase chain reaction. Furthermore, the IFNL3 rs12979860 and the IFNL4 rs368234815 single nucleotide polymorphisms were defined in two of the three patients. RESULTS: All three strains were phylogenetically related to the Russia-derived CRF01 2k/1b while they encompassed the exact same 2k/1b junction site within NS2. CONCLUSION: This is the first report of HCV 2k/1b recombinants in Greece and the greater area of the Balkans.

6.
Hum Gene Ther ; 30(4): 429-445, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30351174

RESUMO

Antigen delivery platforms based on engineered viruses or virus-like particles are currently developed as vaccines against infectious diseases. As the interaction of vaccines with dendritic cells (DCs) shapes the immunological response, we compared the interaction of a range of virus-based vectors and virus-like particles with DCs in a murine model of systemic administration and transcriptome analyses of splenic DCs. The transcriptome profiles of DCs separated the vaccine vectors into two distinct groups characterized by high- and low-magnitude differential gene expression, which strongly correlated with (1) the surface expression of costimulatory molecules CD40, CD83, and CD86 on DCs, and (2) antigen-specific T-cell responses. Pathway analysis using PANOGA (Pathway and Network-Oriented GWAS Analysis) revealed that the JAK/STAT pathway was significantly activated by both groups of vaccines. In contrast, the oxidative phosphorylation pathway was significantly downregulated only by the high-magnitude DC-stimulating vectors. A gene signature including exclusively chemokine-, cytokine-, and receptor-related genes revealed a vector-specific pattern. Overall, this in vivo DC stimulation model demonstrated a strong relationship between the levels of induced DC maturation and the intensity of T-cell-specific immune responses with a distinct cytokine/chemokine profile, metabolic shifting, and cell surface expression of maturation markers. It could represent an important tool for vaccine design.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vetores Genéticos/genética , Fosforilação Oxidativa , Transcrição Gênica , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Biomarcadores , Biologia Computacional/métodos , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Anotação de Sequência Molecular , Baço/citologia , Baço/imunologia , Transcriptoma , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem
7.
Cells ; 7(12)2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30513781

RESUMO

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

8.
Ann Gastroenterol ; 31(5): 593-597, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30174396

RESUMO

BACKGROUND: It has been suggested that hepatitis C virus (HCV) core+1 protein plays a crucial role in the viral life cycle, potentially affecting liver cirrhosis and the development of hepatocellular carcinoma. METHODS: To investigate its relationship with the outcome of HCV standard combination therapy with peginterferon-α plus ribavirin, we screened 139 consecutive HCV patients (119 with chronic HCV infection and 20 who spontaneously cleared HCV) for the presence of anti-core+1 antibodies (Abs). In addition, liver fibrosis was determined by FibroScan in all but one patients. RESULTS: Twenty-nine patients were cirrhotic (stiffness >12.5 kPa, F4 METAVIR), all of them with mild liver cirrhosis (Child-Pugh score A). Eighty-six of 139 patients were treatment-experienced with standard combination therapy. Fifty of them had achieved a sustained virological response, while 36 were non-responders. The prevalence of anti-core+1 Abs in patients with chronic HCV infection was 22.69% (27/119 patients): 18% (9/50 patients) in responders and 36.11% (13/36 patients) in non-responders (P=0.050). Five (17.24%) of the 29 cirrhotic patients and 22 (24.72%) of the 89 non-cirrhotic patients were positive for anti-core+1 Abs (P=0.405). Furthermore, the presence of anti-core+1 Abs correlated with the poor response interleukin (IL) 28B genotype TT (P=0.040). No correlation between spontaneous clearance and anti-core+1 Abs was observed (P=0.088). CONCLUSION: The presence of anti-core+1 Abs might be correlated with the poor response IL28B TT genotype and may negatively affect the outcome of standard combination treatments in HCV patients, suggesting that core+1 may play a biological role in the course of HCV infection.

9.
Sci Rep ; 8(1): 11185, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-30046100

RESUMO

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/ß-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/ß-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several ß-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of ß-catenin nuclear translocation varied in accordance with ß-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/ß-catenin pathway in relevant infection systems.


Assuntos
Carcinoma Hepatocelular/genética , Hepacivirus/genética , Neoplasias Hepáticas/genética , beta Catenina/genética , Transporte Ativo do Núcleo Celular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Genótipo , Células HEK293 , Hepacivirus/patogenicidade , Hepatite C/genética , Hepatite C/patologia , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Fator 1 de Transcrição de Linfócitos T/genética , Via de Sinalização Wnt/genética
10.
J Virol ; 92(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29444947

RESUMO

Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis.IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that immunological responses to core+1/ARFP have been correlated with liver disease severity in chronic HCV patients, we expect that the present work will assist in clarifying the pathophysiological relevance of this protein as a biomarker of disease progression.


Assuntos
Carcinogênese/patologia , Ciclina D1/metabolismo , Hepacivirus/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular , Proliferação de Células/genética , Feminino , Células HEK293 , Hepatite C Crônica/virologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Hepatopatias/virologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fases de Leitura Aberta/genética , Fosforilação , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-vav/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , RNA Mensageiro/genética , Proteínas do Core Viral/genética
11.
J Gen Virol ; 98(7): 1713-1719, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28708052

RESUMO

Hepatitis C virus (HCV) possesses a second open reading frame (ORF) within the core gene encoding an additional protein, known as the alternative reading frame protein (ARFP), F or core+1. The biological significance of the core+1/ARF protein remains elusive. However, several independent studies have shown the presence of core+1/ARFP antibodies in chronically HCV-infected patients. Furthermore, a higher prevalence of core+1/ARFP antibodies was detected in patients with HCV-associated hepatocellular carcinoma (HCC). Here, we investigated the incidence of core+1/ARFPantibodies in chronically HCV-infected patients at different stages of cirrhosis in comparison to chronically HCV-infected patients at earlier stages of disease. Using ELISA, we assessed the prevalence of anti-core+1 antibodies in 30 patients with advanced cirrhosis [model for end-stage liver disease (MELD) ≥15] in comparison with 50 patients with mild cirrhosis (MELD <15) and 164 chronic HCV patients without cirrhosis. 28.7 % of HCV patients with cirrhosis were positive for anti-core+1 antibodies, in contrast with 16.5 % of non-cirrhotic HCV patients. Moreover, there was significantly higher positivity for anti-core+1 antibodies in HCV patients with advanced cirrhosis (36.7 %) compared to those with early cirrhosis (24 %) (P<0.05). These findings, together with the high prevalence of anti-core+1 antibodies in HCV patients with HCC, suggest that core+1 protein may have a role in virus-associated pathogenesis, and provide evidence to suggest that the levels of anti-core+1 antibodies may serve as a marker for disease progression.


Assuntos
Anticorpos Antivirais/imunologia , Carcinoma Hepatocelular/virologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/imunologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Proteínas do Core Viral/imunologia , Adulto , Idoso , Sequência de Bases , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Falência Renal Crônica/imunologia , Falência Renal Crônica/virologia , Cirrose Hepática/complicações , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/imunologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Proteínas do Core Viral/genética
12.
Int J Biochem Cell Biol ; 78: 315-326, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27477312

RESUMO

Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Hepacivirus/fisiologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , MicroRNAs/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica
13.
Int J Mol Sci ; 17(6)2016 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-27304952

RESUMO

Hepatitis C virus (HCV) substantially affects lipid metabolism, and remodeling of sphingolipids appears to be essential for HCV persistence in vitro. The aim of the current study is the evaluation of serum sphingolipid variations during acute HCV infection. We enrolled prospectively 60 consecutive patients with acute HCV infection, most of them already infected with human immunodeficiency virus (HIV), and serum was collected at the time of diagnosis and longitudinally over a six-month period until initiation of antiviral therapy or confirmed spontaneous clearance. Quantification of serum sphingolipids was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spontaneous clearance was observed in 11 out of 60 patients (18.3%), a sustained viral response (SVR) in 43 out of 45 patients (95.5%) receiving an antiviral treatment after follow-up, whereas persistence of HCV occurred in six out of 60 patients (10%). C24-ceramide (C24-Cer)-levels increased at follow-up in patients with spontaneous HCV eradication (p < 0.01), as compared to baseline. Sphingosine and sphinganine values were significantly upregulated in patients unable to clear HCV over time compared to patients with spontaneous clearance of HCV infection on follow-up (p = 0.013 and 0.006, respectively). In summary, the persistence of HCV after acute infection induces a downregulation of C24Cer and a simultaneous elevation of serum sphingosine and sphinganine concentrations.


Assuntos
Ceramidas/metabolismo , Hepacivirus , Hepatite C/metabolismo , Hepatite C/virologia , Esfingosina/análogos & derivados , Esfingosina/sangue , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/metabolismo , Antivirais/uso terapêutico , Biomarcadores , Coinfecção , Feminino , Genótipo , Infecções por HIV , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Carga Viral , Adulto Jovem
14.
PLoS Comput Biol ; 12(3): e1004801, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26998760

RESUMO

Systems biology offers promising approaches for identifying response-specific signatures to vaccination and assessing their predictive value. Here, we designed a modelling strategy aiming to predict the quality of late T-cell responses after vaccination from early transcriptome analysis of dendritic cells. Using standardized staining with tetramer, we first quantified antigen-specific T-cell expansion 5 to 10 days after vaccination with one of a set of 41 different vaccine vectors all expressing the same antigen. Hierarchical clustering of the responses defined sets of high and low T cell response inducers. We then compared these responses with the transcriptome of splenic dendritic cells obtained 6 hours after vaccination with the same vectors and produced a random forest model capable of predicting the quality of the later antigen-specific T-cell expansion. The model also successfully predicted vector classification as low or strong T-cell response inducers of a novel set of vaccine vectors, based on the early transcriptome results obtained from spleen dendritic cells, whole spleen and even peripheral blood mononuclear cells. Finally, our model developed with mouse datasets also accurately predicted vaccine efficacy from literature-mined human datasets.


Assuntos
Células Dendríticas/imunologia , Imunidade Inata/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Transcriptoma/imunologia , Vacinas Virais/imunologia , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Feminino , Imunidade Inata/efeitos dos fármacos , Imunização/métodos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Vacinas Virais/administração & dosagem
16.
Hum Gene Ther Methods ; 26(3): 93-102, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26046494

RESUMO

Dendritic cells (DCs) turn into the most potent antigen-presenting cells following a complex transforming process, which leads to their maturation. Herpes simplex virus-1 (HSV-1) amplicon vectors represent highly versatile viral vector platforms with the ability to transduce immature DCs at exceedingly high efficiencies, while the efficiency of infection of mature DCs is significantly low. However, the bacterial artificial chromosome (BAC)-dependent (BD) amplicon vectors tested so far do not result in the maturation of mouse bone marrow-derived DCs (BMDCs) in vitro. In this study we investigated the effects of light-helper-dependent (LHD) amplicon vectors produced with the replication-defective HSV-1 LaLΔJ helper virus system. First, we observed that transgene expression in BMDC cultures was equally potent between the LHD and the BD amplicon vectors. We determined that the percentage of transduced cells and the duration of transgene expression were negatively influenced by the presence of increasing levels of helper virus. Second, infection by the LHD amplicon vector as well as the helper HSV-1 LaLΔJ virus alone resulted in the phenotypic maturation of BMDCs and the expression of both interferon-stimulated genes and proinflammatory cytokines. Further comparisons of the gene expression of infected DCs showed that while interferon-stimulated genes such as Ifit1, Ifit3, Mx2, Isg15, and Cxcl10 were induced by both BD and LHD amplicon vectors, early proinflammatory cytokine gene expression (Tnfa, Il1a, Il1b, Il6, Il10, Il12b, Cxcl1, and Cxcl16) and DC maturation were mediated only by the LHD amplicons.


Assuntos
Células Dendríticas/citologia , Vírus Auxiliares/genética , Herpesvirus Humano 1/genética , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/metabolismo , Vetores Genéticos/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Transdução Genética , Células Vero
17.
J Virol ; 89(9): 5164-70, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25694591

RESUMO

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.


Assuntos
Expressão Gênica , Hepacivirus/genética , Hepatócitos/virologia , Proteínas do Core Viral/biossíntese , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Replicon , Proteínas do Core Viral/genética
18.
Infect Genet Evol ; 26: 113-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24815730

RESUMO

Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.


Assuntos
Regiões 5' não Traduzidas , Hepacivirus/genética , Hepacivirus/metabolismo , Biossíntese de Proteínas , Proteínas não Estruturais Virais/metabolismo , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Vírus GB C/genética , Vírus GB C/metabolismo , Expressão Gênica , Regulação Viral da Expressão Gênica , Células Hep G2 , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Proteínas não Estruturais Virais/química , eIF-2 Quinase/genética
19.
J Hepatol ; 60(1): 30-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23978712

RESUMO

BACKGROUND & AIMS: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR- and HNF-1α-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. METHODS: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. RESULTS: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1α element in ANGPTL-3 promoter was mutated, while loss of HNF-1α DNA binding to this site was recorded in the presence of HCV core. HNF-1α mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1α-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore core-mediated down-regulation of ANGPTL-3 promoter activity. CONCLUSIONS: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1α binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence.


Assuntos
Angiopoietinas/genética , Hepacivirus/patogenicidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Adulto , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , DNA/metabolismo , Regulação para Baixo , Feminino , Humanos , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Receptores Nucleares Órfãos/fisiologia , Regiões Promotoras Genéticas , Receptores X de Retinoides/fisiologia
20.
J Gen Virol ; 94(Pt 7): 1528-1534, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23580428

RESUMO

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Regulação para Baixo , Hepacivirus/patogenicidade , Fator de Transcrição AP-1/química , Proteínas do Core Viral/metabolismo , Antibacterianos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Hepcidinas , Humanos , Ferro/metabolismo , Fígado/citologia , Regiões Promotoras Genéticas , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Proteínas do Core Viral/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...