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  • 1
    Language: English
    In: Journal of medicinal chemistry, 13 March 2014, Vol.57(5), pp.1627-42
    Description: HCV infections are a major global health burden. After the identification of the virus in 1989, insights into viral replication and drug development have long been hampered by the lack of efficient cell culture models. Their establishment was an important prerequisite for the development of selective antivirals. This review describes the initial difficulties to achieve HCV replication in cell culture, finally leading to the establishment of subgenomic replicons and the infectious virus model (HCVcc). The review further summarizes the current state of HCV cell culture systems with respect to available virus isolates, engineered genomes, and cell types allowing efficient HCV propagation. Finally, we comment on how these cell culture models contributed to the development of directly acting antivirals.
    Keywords: Cell Culture Techniques -- History ; Hepacivirus -- Physiology
    ISSN: 00222623
    E-ISSN: 1520-4804
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  • 2
    Language: English
    In: Gastroenterology, January 2013, Vol.144(1), pp.13-15
    Keywords: Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
    Source: ScienceDirect Journals (Elsevier)
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 30 March 2010, Vol.107(13), pp.5995-9
    Description: Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-alpha-induced NF-kappaB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 muM, except for isosilybin B, which was toxic to cells above 10 muM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 muM. Silymarin suppressed TNF-alpha activation of NF-kappaB dependent transcription, which involved partial inhibition of IkappaB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.
    Keywords: Flavonolignans -- Isolation & Purification ; Liver -- Drug Effects ; Protective Agents -- Isolation & Purification ; Silymarin -- Chemistry
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Gastroenterology
    Description: Dysfunctional CD8+ T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8+ T cells or their surface expression of inhibitory receptors. There is currently no information available about the ability of HCV-specific CD8+ T cells to inhibit viral replication (antiviral efficacy). To analyze the antiviral efficacy of virus-specific CD8+ T cells we utilized an immunological model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8+ T cells from 18 HLA-A*02–positive patients with chronic HCV infection and 15 subjects that resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours co-culture with virus-specific CD8+ T cells. HCV-specific CD8+ T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8+ T cells. Antiviral efficacy was associated with the ability of virus-specific CD8+ T cells to secrete interferon (IFN)-γ. Antiviral efficacy of HCV-specific CD8+ T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin (IL)-2, IL-7, and IL-15 increased the antiviral efficacy of CD127-positive, but not of CD127-negative, HCV-specific CD8+ T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. The ability of CD8+ T cells to inhibit HCV replication is associated with their ability to secrete IFN-γ and their surface expression of CD127 and PD-1.
    Keywords: Immune Response ; Liver Disease ; T Cell Exhaustion ; Antiviral Therapy ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 5
    Language: English
    In: Gastroenterology, 2011, Vol.141(3), pp.1057-1066
    Description: Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of and mouse support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.
    Keywords: Hcv Mouse Model ; Hcv Assembly ; Liver Disease ; Virology ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 6
    Language: English
    In: Gastroenterology, May 2014, Vol.146(5), pp.1361-1372.e9
    Description: Replication of hepatitis C virus (HCV) requires host cell factors, such as cyclophilin A (CypA). CypA binds to HCV’s nonstructural protein (NS)5A to promote replication of viral RNA. CypA antagonists, such as cyclosporines, are potent inhibitors of HCV replication. NS2 modulates sensitivity of HCV to cyclosporines. We investigated why cyclosporines require NS2 to increase their inhibitory effect and how they block HCV replication. We determined replication fitness and sensitivity of various HCV replicons, containing or lacking NS2, to cyclosporine and other direct-acting antiviral agents. We also analyzed the effects of cyclosporine on membranous web formation by electron microscopy. NS2-5B replicons of genotype 2a (JFH1), but not genotype 1b, had increased sensitivity to cyclosporine. This difference was lost with replication-attenuated NS3-5B JFH1 RNAs, showing that cyclosporine sensitivity is linked to reduced replication fitness of NS2-containing HCV RNAs. Fitness also determined sensitivity to a nucleoside analogue and an NS5A inhibitor, but not to telaprevir. Cyclosporine blocked de novo formation of the membranous web, but had little effect on established membranous replication factories. This block was prevented by cyclosporine resistance mutations in NS5A. Cleavage at the NS2/3 junction is a rate-limiting step in replication of particular HCV isolates and determines their sensitivity to CypA inhibitors. These drugs target de novo formation of the membranous web and RNA replication.
    Keywords: Drug Susceptibility ; Direct-Acting Antivirals ; Cyclosporine ; Hcv Protease ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 7
    Article
    Article
    Language: English
    In: Current topics in microbiology and immunology, 2013, Vol.369, pp.167-98
    Description: Genome replication is a crucial step in the life cycle of any virus. HCV is a positive strand RNA virus and requires a set of nonstructural proteins (NS3, 4A, 4B, 5A, and 5B) as well as cis-acting replication elements at the genome termini for amplification of the viral RNA. All nonstructural proteins are tightly associated with membranes derived from the endoplasmic reticulum and induce vesicular membrane alterations designated the membranous web, harboring the viral replication sites. The viral RNA-dependent RNA polymerase NS5B is the key enzyme of RNA synthesis. Structural, biochemical, and reverse genetic studies have revealed important insights into the mode of action of NS5B and the mechanism governing RNA replication. Although a comprehensive understanding of the regulation of RNA synthesis is still missing, a number of important viral and host determinants have been defined. This chapter summarizes our current knowledge on the role of viral and host cell proteins as well as cis-acting replication elements involved in the biogenesis of the membranous web and in viral RNA synthesis.
    Keywords: Transcription, Genetic ; Hepacivirus -- Genetics ; Hepatitis C -- Virology ; RNA, Viral -- Genetics
    ISSN: 0070-217X
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 8
    Language: English
    In: PLoS ONE, 2012, Vol.7(4), p.e36029
    Description: Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication. ; We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles. ; BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs. ; Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture.
    Keywords: Research Article ; Biology ; Medicine ; Virology ; Infectious Diseases ; Gastroenterology And Hepatology
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Gastroenterology, July 2014, Vol.147(1), pp.209-220.e3
    Description: Production of interferon (IFN)-γ by natural killer (NK) cells is attenuated during chronic infection with hepatitis C virus (HCV). We investigated whether this is due to intrinsic or extrinsic mechanisms of NK cells. Peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic HCV infection or uninfected blood donors (controls); NK cells and monocytes were isolated or eliminated. We cultured hepatoma cells that express luciferase-tagged subgenomic HCV replicons (Huh7/HCV replicon cells) or their HCV-negative counterparts (Huh7) with NK cells in the presence or absence of other populations of PBMCs. Antiviral activity, cytotoxicity, and cytokine production were assessed. NK cells produced greater amounts of IFN-γ when PBMC were cocultured with Huh7/HCV replicon cells than with Huh7 cells; NK cells and PBMCs from controls suppressed HCV replication to a greater extent than those from patients with chronic HCV infection. This antiviral effect was predominantly mediated by tumor necrosis factor (TNF)-α and IFN-γ. The antiviral activity of NK cells and their production of IFN-γ were reduced when they were used in coculture alone (rather than with PBMC), or after depletion of CD14 monocytes, after knockdown of the inflammasome in monocytes, or after neutralization of interleukin-18, which is regulated by the inflammasome. These findings indicate a role for monocytes in NK cell activation. Compared with control monocytes, monocytes from patients with chronic HCV infection had reduced TNF-α–mediated (direct) and reduced NK cell–mediated (indirect) antiviral effects. Control monocytes increased the antiviral effects of NK cells from patients with chronic HCV infection and their production of IFN-γ. Monocytes sense cells that contain replicating HCV and respond by producing interleukin-18 via the inflammasome and by activating NK cells. Patients with chronic HCV infection have reduced monocyte function, attenuating NK cell IFN-γ–mediated responses.
    Keywords: Immune Response ; Cytokine Production ; Hepatocyte ; Viral Replication ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 10
    In: PLoS ONE, 2014, Vol.9(5)
    Description: Background & Aims HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication. Methods HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue. Results All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro . HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease. Conclusions Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.
    Keywords: Research Article ; Biology And Life Sciences ; Medicine And Health Sciences
    E-ISSN: 1932-6203
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