Harris H.J., M.J. Farquhar, C.J. Mee, C. Davis, G.M. Reynolds, A. Jennings, K. Hu , F. Yuan, H. Deng, S.G. Hubscher, J.H. Han, P. Balfe and J.A. McKeating J Virol 82:5007-20. 2008.
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Fluorescently tagged AcGFP.CD81 (g.CD81) and DsRed.Claudin1 (r.CLDN1) were investigated with confocal microscopy and fluorescence resonance energy transfer (FRET) analysis in various cell types demonstrating protein association, consistent with the formation of co-receptor complexes. FRET between cell surface expressed g.CD81 (donor) and r.CLDN1 (acceptor) occurred in Huh-7.5, 293T cells transduced to express CLDN1 and T84 colorectal carcinoma cells, suggesting that co-receptor localization and association is not unique to cells which support HCV entry. The transduction of some cell types (Hela, WIF-B) to express exogenous CLDN1 leads to an accumulation of the protein within cells and no detectable cell surface expression, consistent with an inability to support HCVpp entry. However, interpreting data from cells where CLDN1 demonstrates incorrect localization is fraught with difficulties. In contrast, identification of non-permissive cells with native patterns of CLDN1 and CD81 localization, supports the conclusion that co-receptor localization and FRET defined association does not predict cellular permissivity to HCV infection.
Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver derived cells, supporting a model where liver specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI (SR-BI), the tetraspanin CD81 and the tight junction protein Claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as co-receptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques have been developed that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions. AcGFP and DsRed tagged forms of CD81 and CLDN1 co-localize and FRET occurred between the tagged co-receptors at comparable frequencies in permissive and non-permissive cells, consistent with the formation of co-receptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of co-receptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) co-receptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.
Characterization of fluorescently N terminus-tagged CD81 and CLDN1. (A) HepG2 and 293T cells were transduced with retroviral vector pTRIP or pBABE expressing CD81, r.CD81, g.CD81, CLDN1, r.CLDN1, g.CLDN1, CLDN4, or r.CLDN4 and infected with HCVpp-H77, MLVpp, or env-pp. Data are expressed as levels of specific infectivity and represent the mean luciferase levels (relative light units [RLU]) determined from replicate infections, with the Env-pp value subtracted (270 RLU for HepG2 cells and 360 RLU for 293T cells). HepG2 cells expressing r.CD81 (B) or 293T cells expressing r.CLDN1 (C) were stained with antibodies specific for CD81 or CLDN1 respectively. Linear profiling of the fluorescence signal emitted by the tagged protein (black line) and the indirect fluorescence signal from antibody staining (gray line) is shown. The mean fluorescence intensities from fluorescently tagged proteins (r.CD81 and r.CLDN1) and from antibody-stained (anti-CD81 and anti-CLDN1) receptors were obtained by profiling 50 cells. Regions were defined as the nonjunctional Plasma Membrane (PM, black bar), Intracellular (white bar) or Cellular Junctions (CJ, gray bar). All cells were imaged under the same conditions, and the data expressed as arbitrary fluorescence units (F).
Effect of HCV gps on FRET between fluorescently N terminus-tagged CD81 and CLDN1 and viral infectivity. (A) Huh-7.5 cells were incubated with mock or E1-E2 gps at 37°C for 1h and bound protein visualized with anti-E2 1/39 and anti-rat TRITC (red). (B) Huh-7.5 cells expressing g.CD81/r.CLDN1 were incubated with increasing concentrations of E2715 or E1-E2 (0.03 to 1 µM) for 1h at 37°C and fixed in ice-cold methanol, areas of colocalization were selected for FRET analysis. FRET-inferred distances between CD81 and CLDN1 at the nonjunctional PM and CJs were determined. %FRET was unchanged by E2715 or E1-E2 treatments (data not shown); however, E1-E2 reduced the estimateddistance between g.CD81 and r.CLDN1 (**, P + 0.05 [Dunn's test]). (C) Huh-7.5 cells expressing g.CD81/r.CD81 were incubated with a saturatingconcentration (1.0 'M) of E2715 or E1-E2 for 1 h at 37°C and fixed in ice-cold methanol, and areas of colocalization at the nonjunctional PM (black bars) and CJs (gray bars) were selected for FRET analysis. E2715 or E1-E2 treatment(s) had no detectable effect on %FRET and distance(s) between g.CD81 and r.CD81 in comparison to control values. (D) Control or E2715 or E1E2 treated cells were infected for 1h with HCVcc JFH-1, HCVpp-H77, or MLVpp. Unbound virus was removed by washing, and cells incubated for 72h. For HCVcc JFH-1, results are the means from three replicate infections and expressed as relative infectivities compared to the infection of control cells; for HCVpp and MLVpp, specific infectivities are shown and represent the mean luciferase levels (relative light units [RLU]) determined from three replicate infections, with the average Env-pp value subtracted (420 RLU). Incubation with E1-E2 significantly reduced HCVcc and HCVpp relative infectivities (* p < 0.05 - Dunn's test).