RESUMO
BACKGROUND: Hepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model. METHODS: Sub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR. RESULTS: FFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-ß promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response. CONCLUSION: These results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Ácidos Graxos não Esterificados/toxicidade , Hepacivirus/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Interferon-alfa/imunologia , Linhagem Celular , Citofotometria , Ácidos Graxos não Esterificados/análise , Citometria de Fluxo , Hepatócitos/química , Humanos , Luciferases/análise , Microscopia Eletrônica , Oxazinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Coloração e Rotulagem , Estresse FisiológicoRESUMO
The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE-luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.
Assuntos
Expressão Gênica , Hepacivirus/imunologia , Interferon-alfa/imunologia , Receptor de Interferon alfa e beta/biossíntese , Linhagem Celular , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Receptor de Interferon alfa e beta/genética , Deleção de Sequência , Transdução de Sinais , Cultura de VírusRESUMO
BACKGROUND: The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated. RESULTS: Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones. CONCLUSIONS: The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.
Assuntos
Hepacivirus/imunologia , Interferon-alfa/imunologia , Linhagem Celular , Genes Reporter , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepacivirus/classificação , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Janus Quinases/deficiência , Janus Quinases/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição STAT/deficiência , Fatores de Transcrição STAT/imunologia , Coloração e Rotulagem/métodos , Proteínas não Estruturais Virais/genéticaRESUMO
Interferon alpha (IFN-α) binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9) to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD) of either a Stat1 (IRF9-S1C) or Stat2 (IRF9-S2C) protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE) luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.
Assuntos
Hepacivirus/imunologia , Hepacivirus/fisiologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT2/imunologia , Transdução de Sinais , Replicação Viral , Linhagem Celular , Genes Reporter , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Interferon-alfa/imunologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genéticaRESUMO
BACKGROUND: Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone. RESULTS: The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA. CONCLUSION: Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.
Assuntos
Citoplasma/genética , Expressão Gênica , Hepacivirus/fisiologia , Hepatite C/genética , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Proteínas não Estruturais Virais/imunologia , Replicação Viral , Linhagem Celular , Citoplasma/metabolismo , Citoplasma/virologia , Regulação para Baixo , Hepacivirus/enzimologia , Hepacivirus/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Imunoglobulina G/metabolismoRESUMO
PURPOSE: Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients. METHODS: Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting. RESULT: HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rß (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients. CONCLUSION: HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.
Assuntos
Hepacivirus/fisiologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/metabolismo , Autofagia , Transporte Biológico , Biópsia , Células Cultivadas , Regulação para Baixo , Estresse do Retículo Endoplasmático , Etanol/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Fígado/virologia , Cirrose Hepática/patologia , Ribavirina , Replicação ViralRESUMO
AIM: To develop a hepatocellular carcinoma (HCC) xenograft model for studying hepatitis C virus (HCV) replication in a mice, and antiviral treatment. METHODS: We developed a stable S3-green fluorescence protein (GFP) cell line that replicated the GFP-tagged HCV sub-genomic RNA derived from a highly efficient JFH1 virus. S3-GFP replicon cell line was injected subcutaneously into γ-irradiated SCID mice. We showed that the S3-GFP replicon cell line formed human HCC xenografts in SCID mice. Cells were isolated from subcutaneous tumors and then serially passaged multiple times in SCID mice by culturing in growth medium supplemented with G-418. The mouse-adapted S3-GFP replicon cells were implanted subcutaneously and also into the liver of SCID mice via intrasplenic infusion to study the replication of HCV in the HCC xenografts. The tumor model was validated for antiviral testing after intraperitoneal injection of interferon-α (IFN-α). RESULTS: A highly tumorigenic S3-GFP replicon cell line was developed that formed subcutaneous tumors within 2 wk and diffuse liver metastasis within 4 wk in SCID mice. Replication of HCV in the subcutaneous and liver tumors was confirmed by cell colony assay, detection of the viral RNA by ribonuclease protection assay and real-time quantitative reverse transcription polymerase chain reaction. High-level replication of HCV sub-genomic RNA in the tumor could be visualized by GFP expression using fluorescence microscopy. IFN-α cleared HCV RNA replication in the subcutaneous tumors within 2 wk and 4 wk in the liver tumor model. CONCLUSION: A non-infectious mouse model allows us to study replication of HCV in subcutaneous and metastatic liver tumors. Clearance of HCV by IFN-α supports use of this model to test other anti-HCV drugs.
Assuntos
Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Hepacivirus/fisiologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Transplante Heterólogo , Replicação Viral/fisiologia , Animais , Antivirais/uso terapêutico , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite C/tratamento farmacológico , Humanos , Interferon-alfa/farmacologia , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , RNA Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls. CONCLUSIONS: These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.