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Virus-Cell Interactions

CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection

Jie Zhang, Glenn Randall, Adrian Higginbottom, Peter Monk, Charles M. Rice, Jane A. McKeating
Jie Zhang
1Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10021
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Glenn Randall
1Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10021
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Adrian Higginbottom
2School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2RX, United Kingdom
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Peter Monk
2School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2RX, United Kingdom
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Charles M. Rice
1Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10021
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Jane A. McKeating
1Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Diseases, The Rockefeller University, New York, New York 10021
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  • For correspondence: mckeatj@rockefeller.edu
DOI: 10.1128/JVI.78.3.1448-1455.2004
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  • FIG. 1.
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    FIG. 1.

    Anti-CD81 MAb inhibits HCV pseudotype infection of Huh-7.5, Hep3B, and PLC/PR5 cells. Huh-7.5, Hep3B, and PLC/PR5 cells were incubated with anti-CD81 MAb (5A6) or an irrelevant isotype-matched IgG at 5 μg/ml and infected with pseudotypes bearing HCV H or Con1 gp's. Viral stocks were normalized for p24 HIV core antigen and used for infection at 1 ng/well. All infections were performed in triplicate, and the mean luciferase activity (in relative light units [RLU]) is shown. In this and subsequent figures, some standard deviation bars for RLU values are not visible due to compression from the log scale used, but all are within 10%.

  • FIG. 2.
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    FIG. 2.

    siRNA silencing of CD81 expression in Huh-7.5 cells inhibits HCV pseudotype infection. (A) Huh-7.5 cells transfected with either siIRR or siCD81 were stained with IgG1 or anti-CD81 MAb (1.3.3.22) at 54 h posttransfection and analyzed by flow cytometry. (B) siRNA-transfected Huh-7.5 cells were infected with the indicated pseudotypes and analyzed for luciferase activity. (C) siRNA-transfected Huh-7.5 cells were infected similarly with the indicated range of HIV-HCV H pseudotype inocula and analyzed for luciferase activity. All infections were performed in triplicate, and the mean luciferase activity is shown.

  • FIG. 3.
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    FIG. 3.

    Expression of CD81 in HepG2 and HH29 cells confers susceptibility to HCV pseudotype infection. (A) HepG2, HH29, and Huh-7.5 cells alone (upper panels) or after transduction with TRIP-CD81 (lower panels) were stained with an irrelevant IgG and anti-CD81 MAb (1.3.3.22) and analyzed for CD81 expression by flow cytometry. Quadrants were established based on irrelevant isotype-matched IgG staining of the cells. FSC, forward scatter. (B) Transduced (shaded bars) and nontransduced (hatched bars) HepG2, HH29, and Huh-7.5 cells were infected with the indicated pseudotypes 48 h posttransduction and analyzed for luciferase activity. (C) Transduced cells were incubated with either MAbs specific for CD81 (5A6) or an irrelevant isotype-matched IgG at 5 μg/ml and infected with HIV-HCV H E1E2 or with HIV-HCV H E1E2 preincubated with anti-E2 MAb 11/20. All infections were performed in triplicate, and the mean luciferase activity is shown.

  • FIG. 4.
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    FIG. 4.

    The LEL of CD81 is the essential structural component for conferring CD81-dependent susceptibility to HCV pseudotype infection. (A) Schematic representation of CD81 (solid line), CD9 (dotted line), the chimeric molecules CD81-9LEL and CD9-81LEL, and the CD81 mutants with point mutations. (B) HepG2 cells expressing wild-type CD81 (CD81wt) and CD81 variants were infected with the indicated pseudotypes and analyzed for luciferase activity. All infections were performed in triplicate, and the mean luciferase activity is shown. CD9wt, wild-type CD9. (C) Binding of strain H and Con1 soluble E2 to HepG2 cells transduced to express wild-type CD81 and CD81 variants. Results are expressed as median fluorescence intensities. RFU, relative fluorescence units.

Tables

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  • TABLE 1.

    Susceptibility of cells to HCV pseudotype infection

    Cell lineCell typeHuman CD81 expressionaInfectivity (RLU, 103) of pseudotypes expressing:
    No EnvVSV GSF162H E1E2Con1 E1E2
    Hos.CD4.R5Human osteosarcoma24.50.1950.4120.00.10.1
    Huh-7Human hepatoma48.00.14,986.90.868.658.3
    Huh-7.5Human hepatoma81.30.15,469.30.189.363.2
    HepG2Human hepatoma1.90.1890.00.90.10.1
    PLC/PR5Human hepatoma108.00.13,553.30.19.47.5
    HepHHuman liver67.50.1218.70.10.10.1
    HH29Human liver2.20.13,031.70.10.10.1
    Hep3BHuman liver49.40.75,123.60.1120.389.5
    SK Hep1Human liver67.90.23,812.30.10.10.2
    Fet Hep1.3Human liver99.40.22,869.50.10.10.2
    Hepa1-6Mouse liverNA0.1410.70.20.10.1
    H2-35Mouse liverNA0.1232.10.20.20.1
    AML 12Mouse liverNA0.1332.60.10.10.1
    FT0-2BRat liverNA0.1109.10.10.10.1
    PBMCHuman peripheral blood, restingNT0.10.10.10.10.1
    PBMCHuman peripheral blood, phytohemagglutinin activatedNT0.10.966.30.10.1
    MT-2Human T lymphoid95.60.1913.10.10.10.1
    Molt 4Human T lymphoid67.30.23.90.10.10.1
    Hut-78Human T lymphoid171.50.12.60.10.10.1
    DaudiHuman B cell257.30.1589.30.10.10.1
    HeLaHuman epithelial114.40.161.30.10.10.1
    293-THuman embryonal kidney189.40.11,300.10.10.10.1
    SW13Human colorectal adenocarcinoma76.40.1559.20.20.10.1
    Caco-2Human colon adenocarcinoma45.90.1250.30.10.10.1
    HT-29Human fibrosarcomaNT0.11,156.80.10.10.1
    • ↵ a Median fluorescence intensity, in relative fluorescence units. NA, not applicable; NT, not tested.

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CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection
Jie Zhang, Glenn Randall, Adrian Higginbottom, Peter Monk, Charles M. Rice, Jane A. McKeating
Journal of Virology Jan 2004, 78 (3) 1448-1455; DOI: 10.1128/JVI.78.3.1448-1455.2004

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CD81 Is Required for Hepatitis C Virus Glycoprotein-Mediated Viral Infection
Jie Zhang, Glenn Randall, Adrian Higginbottom, Peter Monk, Charles M. Rice, Jane A. McKeating
Journal of Virology Jan 2004, 78 (3) 1448-1455; DOI: 10.1128/JVI.78.3.1448-1455.2004
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KEYWORDS

Antigens, CD
hepacivirus
membrane proteins
Receptors, Virus
Viral Envelope Proteins
viral structural proteins

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