Persistence of HCV in Quiescent Hepatic Cells Under Conditions of an Interferon-Induced Antiviral Response

doi:10.1053/j.gastro.2012.04.018

Gastroenterology

Volume 143, Issue 2, August 2012, Pages 429-438.e8

https://doi.org/10.1053/j.gastro.2012.04.018 Get rights and content

Background & Aims

Hepatitis C virus (HCV) is a common cause of chronic liver disease. Many patients do not clear the viral infection; little is known about the mechanisms of HCV persistence or the frequent failure of interferon (IFN) to eliminate it. Better culture systems are needed to study viral replication in quiescent liver cells.

Methods

We used human hepatoma (Huh7.5) cells and those that had undergone proliferation arrest and differentiation (Huh7.5^dif) to study the persistence of HCV infection following exposure of the cells to IFN-α and to compare the antiviral effects of IFN-α and IFN-λ. We validated these results with primary human hepatocytes and Huh7 cells that expressed an IFN-inducible fluorophore.

Results

Following infection of Huh7.5^dif cells, HCV replicated persistently and released infectious particles. Long-term exposure of the cells to IFN-α reduced HCV replication ∼1000-fold but did not eliminate the virus; viral replication rebounded after withdrawal of IFN, as it does in patients with chronic HCV infection. HCV replicated at higher levels, but not exclusively, in cells that had a low level of response to IFN-α. Following incubation of cells with equipotent concentrations of IFN-α or IFN-λ, Huh7.5^dif cells expressed a wider pattern of IFN-stimulated genes than undifferentiated Huh7.5 cells or primary human hepatocytes, indicating that the antiviral response depends on the differentiation status of the cells.

Conclusions

We developed a cell culture system using hepatoma cells to study persistent HCV infection during the type I or type III IFN-induced antiviral response. The level and range of the antiviral responses were associated with the differentiation status of the cells. We propose that HCV exploits the stochastic nature of the response of hepatocytes to IFN to sustain persistence.

Section snippets

Virus Production and Measurement of Infectivity Titers

Virus stocks were obtained by transfecting Huh7.5 cells (kindly provided by C. M. Rice, Rockefeller University, New York, NY) with in vitro transcripts of the intergenotypic Jc1 HCV genome.¹⁶ Infectivity titers in culture supernatants and virus stocks were quantified by determining the tissue culture infectious dose 50 (TICD₅₀) per milliliter using a limiting dilution assay as described elsewhere.¹⁷ Further details are given in Supplementary Materials and Methods.

Differentiation of Huh7.5 Cells and IFN Treatment

Cells were differentiated by

HCV Replication and Virus Production in Growth-Arrested and Differentiated Huh7.5 Cells

To study persistent HCV replication in long-term quiescent and differentiated cells, we adapted a recently described protocol¹⁵ to Huh7.5 cells and first determined permissiveness and long-term viral replication. Cells were seeded at low density and cultured for 14 days in medium containing 1% DMSO. At the end of this incubation period, cells stopped growth and no signs of mitoses were detected (not shown). Microscopic inspection revealed that cells grew in ridge-like structures composed of

Discussion

In this study, we show persistent HCV replication in differentiated and quiescent Huh7.5 cells that support HCV replication and virus production for at least 1 month, which is in agreement with an earlier report.¹⁵ This result is remarkable, because previous studies using the replicon system observed a tight coupling between HCV RNA replication and cell growth.²⁵ The coupling might be linked to intracellular NTP pools that are reduced in quiescent cells concomitant with a massive reduction of

Acknowledgments

The authors thank Ulrike Herian for excellent technical assistance; Ina Poser and Frank Buchholz for initial advice in setting up the BAC system; Monika Langlotz and the FACS Facility at the Center for Molecular Biology Heidelberg; Thomas Longerich for preparation of the liver samples; Marco Binder and Volker Lohmann for critical discussion of the project; Charles M. Rice, Ilka Julkunen, Frank Buchholz, Andriy Khobta and Georg Kochs for providing reagents; and the Genomics and Proteomics Core

References (36)

I.M. Jacobson et al.
Prevalence and challenges of liver diseases in patients with chronic hepatitis C virus infection
Clin Gastroenterol Hepatol
(2010)
A. Shavinskaya et al.
The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly
J Biol Chem
(2007)
J.M. Vrolijk et al.
A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C
J Virol Methods
(2003)
E. Ford et al.
The transcriptional code of human IFN-beta gene expression
Biochim Biophys Acta
(2010)
K. Murakami et al.
Production of infectious hepatitis C virus particles in three-dimensional cultures of the cell line carrying the genome-length dicistronic viral RNA of genotype 1b
Virology
(2006)
J. Vermehren et al.
New HCV therapies on the horizon
Clin Microbiol Infect
(2011)
G. Cabibbo et al.
Epidemiology, risk factors and surveillance of hepatocellular carcinoma
Eur Rev Med Pharmacol Sci
(2010)
P. Ferenci et al.
Impact of HCV protease-inhibitor-based triple therapy for chronic HCV genotype 1 infection
Antivir Ther
(2011)
D.L. Thomas et al.
Genetic variation in IL28B and spontaneous clearance of hepatitis C virus
Nature
(2009)
A. Ruiz-Extremera et al.
Genetic variation in interleukin 28B with respect to vertical transmission of hepatitis C virus and spontaneous clearance in HCV-infected children
Hepatology
(2011)

A. Rauch et al.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study

Gastroenterology

(2010)

D. Ge et al.

Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance

Nature

(2009)

R. Thimme et al.

Failure of innate and adaptive immune responses in controlling hepatitis C virus infection

FEMS Microbiol Rev

(2012)

C.B. Bigger et al.

Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees

J Virol

(2004)

M. Sarasin-Filipowicz et al.

Interferon signaling and treatment outcome in chronic hepatitis C

Proc Natl Acad Sci U S A

(2008)

M. Frese et al.

Interferon-alpha inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway

J Gen Virol

(2001)

I.W. Cheney et al.

Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha, beta, and gamma interferons

J Virol

(2002)

J. Larkin et al.

Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system

J Interferon Cytokine Res

(2003)

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by the Bundesministerium für Bildung und Forschung (01KI1008A), the Deutsche Forschungsgemeinschaft (FOR1202, TP1), the EU (SysPatho; grant agreement no. 260429), and the Schweizerische Nationalfonds (SNF #PA00P3_124161).

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Original ResearchBasic and Translational—LiverPersistence of HCV in Quiescent Hepatic Cells Under Conditions of an Interferon-Induced Antiviral Response

Background & Aims

Methods

Results

Conclusions

Section snippets

Virus Production and Measurement of Infectivity Titers

Differentiation of Huh7.5 Cells and IFN Treatment

HCV Replication and Virus Production in Growth-Arrested and Differentiated Huh7.5 Cells

Discussion

Acknowledgments

Clin Gastroenterol Hepatol

J Biol Chem

J Virol Methods

Biochim Biophys Acta

Virology

Clin Microbiol Infect

Epidemiology, risk factors and surveillance of hepatocellular carcinoma

Eur Rev Med Pharmacol Sci

Impact of HCV protease-inhibitor-based triple therapy for chronic HCV genotype 1 infection

Antivir Ther

Genetic variation in IL28B and spontaneous clearance of hepatitis C virus

Nature

Genetic variation in interleukin 28B with respect to vertical transmission of hepatitis C virus and spontaneous clearance in HCV-infected children

Hepatology

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study

Gastroenterology

Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance

Nature

Failure of innate and adaptive immune responses in controlling hepatitis C virus infection

FEMS Microbiol Rev

Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees

J Virol

Interferon signaling and treatment outcome in chronic hepatitis C

Proc Natl Acad Sci U S A

Interferon-alpha inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway

J Gen Virol

Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha, beta, and gamma interferons

J Virol

Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system

J Interferon Cytokine Res

Original Research
Basic and Translational—Liver
Persistence of HCV in Quiescent Hepatic Cells Under Conditions of an Interferon-Induced Antiviral Response