RESUMO
BACKGROUND: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes. METHODS: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-ß1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber. RESULTS: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner. CONCLUSIONS: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Evasão da Resposta Imune , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Antígenos de Diferenciação/imunologia , Regulação da Expressão Gênica/imunologia , Hepatite C/patologia , Humanos , Fígado/patologia , Fígado/virologia , Linfócitos T Reguladores/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0070809.].
RESUMO
Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.
Assuntos
Vírus da Hepatite E/patogenicidade , Proteínas Virais/química , Proteínas Virais/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Glicosilação , Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Mutação , Sinais Direcionadores de Proteínas , Estabilidade Proteica , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The hepatitis E virus (HEV) genome is a single-stranded, positive-sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose-dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA-resistant GBF1 mutant rescuing HEV replication in BFA-treated cells, confirmed that GBF1 is the only BFA-sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1-regulated mechanisms.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus da Hepatite E/crescimento & desenvolvimento , RNA Viral/biossíntese , Replicação Viral/fisiologia , Antivirais/farmacologia , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Hepatite E/patologia , Hepatite E/virologia , Vírus da Hepatite E/genética , Humanos , Piridinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.
Assuntos
Proteínas do Capsídeo/isolamento & purificação , Vírus da Hepatite E/fisiologia , Hepatite E/metabolismo , Proteínas Virais/isolamento & purificação , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Hepatite E/etiologia , Hepatite E/patologia , Hepatócitos , Humanos , CamundongosRESUMO
OBJECTIVE: The hepatitis E virus (HEV) is responsible for approximately 20 million infections per year worldwide. Although most infected people can spontaneously clear an HEV infection, immune-compromised individuals may evolve towards chronicity. Chronic HEV infection can be cured using ribavirin, but viral isolates with low ribavirin sensitivity have recently been identified. Although some HEV isolates can be cultured in vitro, in vivo studies are essentially limited to primates and pigs. Since the use of these animals is hampered by financial, practical and/or ethical concerns, we evaluated if human liver chimeric mice could serve as an alternative. DESIGN: Humanised mice were inoculated with different HEV-containing preparations. RESULTS: Chronic HEV infection was observed after intrasplenic injection of cell culture-derived HEV, a filtered chimpanzee stool suspension and a patient-derived stool suspension. The viral load was significantly higher in the stool compared with the plasma. Overall, the viral titre in genotype 3-infected mice was lower than that in genotype 1-infected mice. Analysis of liver tissue of infected mice showed the presence of viral RNA and protein, and alterations in host gene expression. Intrasplenic injection of HEV-positive patient plasma and oral inoculation of filtered stool suspensions did not result in robust infection. Finally, we validated our model for the evaluation of novel antiviral compounds against HEV using ribavirin. CONCLUSIONS: Human liver chimeric mice can be infected with HEV of different genotypes. This small animal model will be a valuable tool for the in vivo study of HEV infection and the evaluation of novel antiviral molecules.
Assuntos
Modelos Animais de Doenças , Vírus da Hepatite E/genética , Hepatite E/virologia , Fígado/química , RNA Viral/análise , Proteínas Virais/análise , Animais , Antivirais/uso terapêutico , Expressão Gênica , Genótipo , Hepatite E/tratamento farmacológico , Hepatite E/genética , Hepatócitos/transplante , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Ribavirina/uso terapêutico , Quimeras de Transplante , Carga ViralRESUMO
UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.
Assuntos
Aminoquinolinas/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Aminoquinolinas/química , Aminoquinolinas/isolamento & purificação , Animais , Antivirais/química , Antivirais/isolamento & purificação , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral , Hepatite C/tratamento farmacológico , Hepatócitos/virologia , Humanos , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Mutação de Sentido Incorreto , Genética Reversa , Resultado do Tratamento , Proteínas do Envelope Viral/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
UNLABELLED: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. IMPORTANCE: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Polifenóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antocianinas/administração & dosagem , Antocianinas/farmacologia , Antivirais/administração & dosagem , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular , Microscopia Crioeletrônica , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Hepacivirus/ultraestrutura , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Interferon-alfa/administração & dosagem , Polifenóis/administração & dosagem , Prolina/administração & dosagem , Prolina/análogos & derivadosRESUMO
UNLABELLED: In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCE: Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.
Assuntos
Hepacivirus/fisiologia , Ionóforos/farmacologia , Monensin/farmacologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Farmacorresistência Viral/genética , Técnica Indireta de Fluorescência para Anticorpo , Hepacivirus/genética , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Mutação de Sentido Incorreto/genética , Testes de Neutralização , Proteínas Virais/metabolismoRESUMO
Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.
Assuntos
Regulação Viral da Expressão Gênica , Hepacivirus/fisiologia , Proteínas do Core Viral/biossíntese , Replicação Viral , Linhagem Celular , Perfilação da Expressão Gênica , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Estabilidade Proteica , Fatores de Tempo , Proteínas do Core Viral/genéticaRESUMO
Hepatitis C virus (HCV) infection is a worldwide health problem. Vaccines against this pathogen are not available and advances in this field are limited because of the high genetic variability of the virus, inaccessibility of animal models, and incomplete definition of immunological correlates of protection. In the present work, a chimeric protein, Eq1, encompassing HCV amino acid regions from structural antigens, was generated. Eq1 was expressed in GC-366 bacterial cells. After cell disruption, Eq1 was purified from the insoluble fraction by sequential steps of differential solubilization and metal chelating affinity chromatography. Eq1 was specifically recognized by anti-HCV positive human sera. Moreover, immunization of BALB/c mice with different doses of Eq1 formulated either in Alum or Freund's incomplete adjuvant elicited both humoral- and cellular-specific immune responses. Doses of 20 µg of Eq1 induced the strongest cell-mediated immune responses and only the formulation of this dose in Alum elicited a neutralizing antibody response against heterologous cell culture HCV. All these data together indicate that Eq1 is immunogenic in mice and might be an interesting component of vaccine candidates against HCV infection.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Imunidade Celular , Imunidade Humoral , Proteínas Recombinantes de Fusão/imunologia , Animais , Epitopos/imunologia , Adjuvante de Freund , Hepatite C/prevenção & controle , Humanos , Lipídeos , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
AIM: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. METHODS: CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. RESULTS: From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CONCLUSION: CIGB-230, combined with IFN-α-based therapy, modifies the immune response in chronic patients. The study provides evidence for the design of more effective therapeutic vaccine interventions against HCV.
Assuntos
Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Ribavirina/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Adulto , Antivirais/efeitos adversos , Biomarcadores/sangue , Células Cultivadas , Cuba , Método Duplo-Cego , Esquema de Medicação , Quimioterapia Combinada , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Esquemas de Imunização , Interferon alfa-2 , Interferon-alfa/efeitos adversos , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Ribavirina/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Vacinas de DNA/efeitos adversos , Vacinas contra Hepatite Viral/efeitos adversosRESUMO
Recent reports indicate that the replication of hepatitis C virus (HCV) depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA), which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.
Assuntos
Brefeldina A/farmacologia , Carcinoma Hepatocelular/virologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Complexo de Golgi/metabolismo , Hepacivirus/fisiologia , Neoplasias Hepáticas/virologia , Replicação Viral/efeitos dos fármacos , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Apolipoproteínas E/metabolismo , Sequência de Bases , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Compartimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Complexo de Golgi/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hepatite C/patologia , Hepatite C/virologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de ProteínaRESUMO
Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells' permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.
Assuntos
Hepacivirus/genética , Adaptação Fisiológica/genética , Carcinoma Hepatocelular , Análise Mutacional de DNA , Células Hep G2 , Hepacivirus/imunologia , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Mutação , Cultura Primária de Células , RNA Viral/genética , Carga Viral , Cultura de Vírus , Internalização do VírusRESUMO
BACKGROUND: The envelope glycoprotein E2 of hepatitis C virus (HCV) contains several hypervariable regions. Interestingly, 2 regions of intragenotypic hypervariability within E2 have been described as being specific to HCV subtype 3a. Based on their amino acid position in E2, they were named HVR495 and HVR575. Here, we further investigated these regions in order to better understand their role in HCV infection. METHODS: Sequences of HCV envelope glycoproteins from Pakistani patients infected with subtype 3a were cloned and compared with other subtype 3a sequences. The entry functions and the sensitivity to antibody neutralization of selected HCV glycoprotein sequences were tested in the HCV pseudotyped particles (HCVpp) system. In addition, the cell-cultured HCV system (HCVcc) was also used to confirm some of the data obtained with the HCVpp system. RESULTS: We observed interesting new features within HVR495 and HVR575 for several subtype 3a isolates. Indeed, changes in glycosylation sites were observed with the appearance of a new glycosylation site within HVR495. Importantly, HCVpp and HCVcc that contained this new HVR495 glycosylation site were less sensitive to antibody neutralization. CONCLUSIONS: We identified a new glycosylation site within the HVR495 region of HCV subtype 3a that has a protective effect against antibody neutralization.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C/virologia , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Linhagem Celular , Glicosilação , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/imunologia , Humanos , Mutação , Paquistão , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Tetraspanina 28/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
UNLABELLED: Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. CONCLUSION: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals.
Assuntos
Aminoquinolinas/farmacologia , Antivirais/uso terapêutico , Compostos Ferrosos/farmacologia , Hepacivirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Hepacivirus/genética , Hepatite C/prevenção & controle , Humanos , Interferon-alfa/administração & dosagem , Metalocenos , Prolina/administração & dosagem , Prolina/análogos & derivados , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.
Assuntos
Hepacivirus/genética , Hepatite C/virologia , Seleção Genética , Proteínas do Envelope Viral/genética , Montagem de Vírus , Substituição de Aminoácidos , Linhagem Celular , Teste de Complementação Genética , Hepacivirus/fisiologia , Humanos , Mutação , RNA Viral/genética , Replicon , Genética Reversa , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
UNLABELLED: Here, we identify (-)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 µM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. CONCLUSION: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte.
Assuntos
Antivirais/farmacologia , Catequina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Hepatócitos/virologia , Extratos Vegetais/farmacologia , Chá , Internalização do Vírus/efeitos dos fármacos , Animais , Catequina/farmacologia , Bovinos , Linhagem Celular , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C/patologia , Hepatite C/fisiopatologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Rim/virologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Modelos Animais , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which contain a spheroid perinuclear inclusion body, was found in our cellular clone. In conclusion, our data suggest that Huh-7w7.3 cells constitute an excellent model for determining the cellular factor(s) involved in the process of spheroid perinuclear body formation.
Assuntos
Citoplasma/metabolismo , Hepatócitos/patologia , Queratinas/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Clonais/patologia , Humanos , Queratinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA(+/+)-SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.