Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line

ABSTRACT The Hepatitis C virus (HCV), a member of the family Flaviviridae, is a major cause of chronic liver disease. Patients are currently treated with alpha interferon (IFN-α) that is given alone or in combination with ribavirin. Unfortunately, this treatment is ineffective in eliminating the virus in a large proportion of individuals. IFN-induced antiviral activities have been intensively studied in the HCV replicon system. It was found that both IFN-α and IFN-γ inhibit HCV replicons, but the underlying mechanisms have not yet been identified. Of note is that nearly all of these studies were performed with the human hepatoma cell line Huh-7. Here, we report that genotypes 1b and 2a replicons also replicate in the human hepatoblastoma cell line HuH6. Similar to what has been described for Huh-7 cells, we observed that efficient HCV replication in HuH6 cells depends on the presence of cell culture-adaptive mutations and the permissiveness of the host cell. However, three major differences exist: in HuH6 cells, viral replication is (i) independent from ongoing cell proliferation, (ii) less sensitive to certain antiviral compounds, and (iii) highly resistant to IFN-γ. The latter is not due to a general defect in IFN signaling, as IFN-γ induces the nuclear translocation of signal transducer and activator of transcription 1 (STAT1), the enhanced transcription of several IFN-regulated genes, and the inhibition of unrelated viruses such as influenza A virus and Semliki Forest virus. Taken together, the results establish HuH6 replicon cells as a valuable tool for IFN studies and for the evaluation of antiviral compounds.

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Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.77.5.3007-3019.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3007-3019 ◽

Cited By ~ 304

Author(s):

Volker Lohmann ◽

Sandra Hoffmann ◽

Ulrike Herian ◽

Francois Penin ◽

Ralf Bartenschlager

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Host Cell ◽

Cell Line ◽

Rna Replication ◽

Strong Increase ◽

Hepatoma Cell Line ◽

Replication Efficiency ◽

Adaptive Mutations

ABSTRACT Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.

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Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

Viruses ◽

10.3390/v12010036 ◽

2019 ◽

Vol 12 (1) ◽

pp. 36

Author(s):

Christopher Dächert ◽

Evgeny Gladilin ◽

Marco Binder

Keyword(s):

Gene Expression ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Gene Expression Profiling ◽

Cell Line ◽

Expression Profiling ◽

Hepatoma Cell ◽

Host Factors ◽

Hepatoma Cell Line ◽

Huh7 Cells

Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

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Efficient replication systems for hepatitis C virus using a new human hepatoma cell line

Virus Research ◽

10.1016/j.virusres.2009.08.006 ◽

2009 ◽

Vol 146 (1-2) ◽

pp. 41-50 ◽

Cited By ~ 38

Author(s):

Nobuyuki Kato ◽

Kyoko Mori ◽

Ken-ichi Abe ◽

Hiromichi Dansako ◽

Misao Kuroki ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Human Hepatoma ◽

Human Hepatoma Cell ◽

Human Hepatoma Cell Line ◽

Replication Systems ◽

Efficient Replication

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Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.76.24.13001-13014.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 13001-13014 ◽

Cited By ~ 867

Author(s):

Keril J. Blight ◽

Jane A. McKeating ◽

Charles M. Rice

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hepatoma Cell ◽

Single Amino Acid ◽

Prolonged Treatment ◽

Hepatoma Cell Line ◽

Acceptor Site ◽

Adaptive Mutations ◽

Cellular Environment

ABSTRACT Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

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