Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor

ABSTRACT CD81 has been described as a putative receptor for hepatitis C virus (HCV); however, its role in HCV cell entry has not been characterized due to the lack of an efficient cell culture system. We have examined the role of CD81 in HCV glycoprotein-dependent entry by using a recently developed retroviral pseudotyping system. Human immunodeficiency virus (HIV) pseudotypes bearing HCV E1E2 glycoproteins show a restricted tropism for human liver cell lines. Although all of the permissive cell lines express CD81, CD81 expression alone is not sufficient to allow viral entry. CD81 is required for HIV-HCV pseudotype infection since (i) a monoclonal antibody specific for CD81 inhibited infection of susceptible target cells and (ii) silencing of CD81 expression in Huh-7.5 hepatoma cells by small interfering RNAs inhibited HIV-HCV pseudotype infection. Furthermore, expression of CD81 in human liver cells that were previously resistant to infection, HepG2 and HH29, conferred permissivity of HCV pseudotype infection. The characterization of chimeric CD9/CD81 molecules confirmed that the large extracellular loop of CD81 is a determinant for viral entry. These data suggest a functional role for CD81 as a coreceptor for HCV glycoprotein-dependent viral cell entry.

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Parathyroid hormone-related peptide is expressed and rapidly inducible in human liver cell cultures that have a bile duct phenotype

Journal of Hepatology ◽

10.1016/0168-8278(95)80330-0 ◽

1995 ◽

Vol 23 (2) ◽

pp. 160-165 ◽

Cited By ~ 15

Author(s):

Tania Roskams ◽

Han Moshage ◽

Eric Depla ◽

Marc Willems ◽

Valeer Desmet ◽

...

Keyword(s):

Parathyroid Hormone ◽

Bile Duct ◽

Cell Cultures ◽

Liver Cell ◽

Human Liver ◽

Related Peptide ◽

Human Liver Cell ◽

Parathyroid Hormone Related Peptide

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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 ◽

10.1016/j.virol.2006.03.038 ◽

2006 ◽

Vol 351 (2) ◽

pp. 381-392 ◽

Cited By ~ 33

Author(s):

Kyoko Murakami ◽

Koji Ishii ◽

Yousuke Ishihara ◽

Sayaka Yoshizaki ◽

Keiko Tanaka ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Three Dimensional ◽

Infectious Hepatitis ◽

Viral Rna ◽

Genome Length ◽

Virus Particles ◽

Genotype 1B

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Preclinical assessment of hepatocyte-targeted MR contrast agents in stable human liver cell cultures

Journal of Magnetic Resonance Imaging ◽

10.1002/jmri.1880080326 ◽

1998 ◽

Vol 8 (3) ◽

pp. 687-689 ◽

Cited By ~ 8

Author(s):

Peter Reimer ◽

Augustinus Bader ◽

Ralph Weissleder

Keyword(s):

Contrast Agents ◽

Cell Cultures ◽

Liver Cell ◽

Human Liver ◽

Mr Contrast Agents ◽

Human Liver Cell

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654 PRODUCTION OF INFECTIOUS HEPATITIS C VIRUS IN A PRIMARY CULTURE OF HUMAN LIVER SLICES

Journal of Hepatology ◽

10.1016/s0168-8278(10)60656-3 ◽

2010 ◽

Vol 52 ◽

pp. S255

Author(s):

S. Lagaye ◽

H. Shen ◽

J. Gaston ◽

M. Nascimbeni ◽

M. Triyatni ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Primary Culture ◽

Human Liver ◽

Infectious Hepatitis ◽

Liver Slices ◽

Human Liver Slices

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Direct visualization of hepatitis C virus-infected Huh7.5 cells with a high titre of infectious chimeric JFH1-EGFP reporter virus in three-dimensional Matrigel cell cultures

Journal of General Virology ◽

10.1099/vir.0.055772-0 ◽

2014 ◽

Vol 95 (2) ◽

pp. 423-433 ◽

Cited By ~ 11

Author(s):

Shuanghu Liu ◽

Ren Chen ◽

Curt H. Hagedorn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Cultures ◽

Three Dimensional ◽

Egfp Expression ◽

Reporter Virus ◽

Egfp Gene ◽

3D Cultures ◽

Egfp Reporter ◽

Ns5a Region

Identification of the hepatitis C virus (HCV) JFH1 isolate enabled the development of infectious HCV cell culture systems. However, the relatively low virus titres and instability of some chimeric JFH1 reporter viruses restricts some uses of this system. We describe a higher-titre JFH1-EGFP reporter virus where the NS5A V3 region was replaced with the EGFP gene and adapted by serial passage in Huh7.5 cells. Six adaptive mutants were identified: one each in E2, P7 and NS4B, plus three in the NS5A region. These adaptive mutants increased the reporter virus titres to 1×106 immunofluorescent focus-forming units ml−1, which is the highest titre of JFH1-EGFP reporter virus reported to our knowledge. This chimeric virus did not lose EGFP expression following 40 days of passage and it can be used to test the activity of HCV antivirals by measuring EGFP fluorescence in 96-well plates. Moreover, this reporter virus allows living infected Huh7.5 cells in Matrigel three-dimensional (3D) cultures to be visualized and produces infectious viral particles in these 3D cultures. The chimeric NS5A-EGFP infectious JFH1 reporter virus described should enable new studies of the HCV life cycle in 3D cell cultures and will be useful in identifying antivirals that interfere with HCV release or entry.

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