Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

ABSTRACT To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients—β-carotene, vitamin D2, and linoleic acid—inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, β-carotene, vitamin D2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

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Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

Journal of General Virology ◽

10.1099/vir.0.040725-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1422-1431 ◽

Cited By ~ 8

Author(s):

Midori Takeda ◽

Masanori Ikeda ◽

Yasuo Ariumi ◽

Takaji Wakita ◽

Nobuyuki Kato

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hepatoma Cell ◽

Rna Replication ◽

Hcv Rna ◽

Reporter Assay ◽

Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.78.23.13306-13314.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13306-13314 ◽

Cited By ~ 102

Author(s):

Petra Neddermann ◽

Manuela Quintavalle ◽

Chiara Di Pietro ◽

Angelica Clementi ◽

Mauro Cerretani ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Kinase Inhibitors ◽

Rna Replication ◽

Hcv Rna ◽

Adaptive Mutations ◽

Direct Demonstration ◽

Huh7 Cells ◽

Efficient Replication

ABSTRACT Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.

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Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Journal of Virology ◽

10.1128/jvi.77.5.3181-3190.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3181-3190 ◽

Cited By ~ 258

Author(s):

Keril J. Blight ◽

Jane A. McKeating ◽

Joseph Marcotrigiano ◽

Charles M. Rice

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Full Length ◽

Hcv Rna ◽

Metabolic Labeling ◽

Virus Genotype ◽

Adaptive Mutations ◽

Genotype 1A

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

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Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

Journal of Virology ◽

10.1128/jvi.00526-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10999-11009 ◽

Cited By ~ 149

Author(s):

Pablo Gastaminza ◽

Kelly A. Dryden ◽

Bryan Boyd ◽

Malcolm R. Wood ◽

Mansun Law ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Structural Characteristics ◽

Buoyant Density ◽

Hcv Rna ◽

Membrane Bilayer ◽

Biophysical Characterization ◽

Negative Stain ◽

A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I

Journal of Virology ◽

10.1128/jvi.79.5.2689-2699.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2689-2699 ◽

Cited By ~ 641

Author(s):

Rhea Sumpter ◽

Yueh-Ming Loo ◽

Eileen Foy ◽

Kui Li ◽

Mitsutoshi Yoneyama ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Infections ◽

Cultured Cells ◽

Rna Replication ◽

Rna Helicase ◽

Hcv Rna ◽

Helicase Domain ◽

Replication Studies ◽

Virus Rna

ABSTRACT Virus-responsive signaling pathways that induce alpha/beta interferon production and engage intracellular immune defenses influence the outcome of many viral infections. The processes that trigger these defenses and their effect upon host permissiveness for specific viral pathogens are not well understood. We show that structured hepatitis C virus (HCV) genomic RNA activates interferon regulatory factor 3 (IRF3), thereby inducing interferon in cultured cells. This response is absent in cells selected for permissiveness for HCV RNA replication. Studies including genetic complementation revealed that permissiveness is due to mutational inactivation of RIG-I, an interferon-inducible cellular DExD/H box RNA helicase. Its helicase domain binds HCV RNA and transduces the activation signal for IRF3 by its caspase recruiting domain homolog. RIG-I is thus a pathogen receptor that regulates cellular permissiveness to HCV replication and, as an interferon-responsive gene, may play a key role in interferon-based therapies for the treatment of HCV infection.

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An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

Journal of Virology ◽

10.1128/jvi.78.20.11393-11400.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11393-11400 ◽

Cited By ~ 112

Author(s):

Menashe Elazar ◽

Ping Liu ◽

Charles M. Rice ◽

Jeffrey S. Glenn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hcv Rna ◽

Membrane Association ◽

Amphipathic Helix ◽

Nonstructural Proteins ◽

Replication Complex ◽

Cytoplasmic Membranes ◽

Positive Strand Rna

ABSTRACT Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

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Synthesis and SAR of piperazinyl-N-phenylbenzamides as inhibitors of hepatitis C virus RNA replication in cell culture

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2009.01.066 ◽

2009 ◽

Vol 19 (6) ◽

pp. 1779-1783 ◽

Cited By ~ 25

Author(s):

Immacolata Conte ◽

Claudio Giuliano ◽

Caterina Ercolani ◽

Frank Narjes ◽

Uwe Koch ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Replication ◽

Hepatitis C Virus Rna ◽

Virus Rna

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Novel Robust Hepatitis C Virus Mouse Efficacy Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00413-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3260-3268 ◽

Cited By ~ 22

Author(s):

Qing Zhu ◽

Yoko Oei ◽

Dirk B. Mendel ◽

Evelyn N. Garrett ◽

Montesa B. Patawaran ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Small Animal ◽

Rna Replication ◽

Treatment Withdrawal ◽

Whole Body ◽

Hcv Rna ◽

Whole Body Imaging

ABSTRACT The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

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Visualization of Double-Stranded RNA in Cells Supporting Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.01565-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2182-2195 ◽

Cited By ~ 126

Author(s):

Paul Targett-Adams ◽

Steeve Boulant ◽

John McLauchlan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Three Dimensional ◽

Rna Replication ◽

Hcv Rna ◽

Double Stranded Rna ◽

Core Proteins ◽

Virus Rna ◽

Infected Cells

ABSTRACT The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.

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