Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes

Abstract

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.

FIG. 1.

Alignment of amino acid sequences of tupaia, mouse, and human SR-BI. Tupaia SR-BI cDNA was cloned and sequenced by RT-PCR of tupaia mRNA with human SR-BI-specific primers as described in Materials and Methods. SR-BI amino acid sequences of mouse and human SR-BI are depicted according to a previous report (1). Amino acid homology and differences between species are indicated by different colors.

FIG. 2.

Expression of SR-BI in PTH and human hepatoma cells. (A) PTH, human hepatoma HepG2 cells, and Sf9 insect cell lysates were subjected to SDS-PAGE. Following gel transfer to polyvinylidene difluoride membranes, immunoblotting was performed using rabbit anti-SR-BI polyclonal antibody (NB 400-104) and horseradish peroxidase-conjugated anti-rabbit IgG. The presence of SR-BI is indicated on the left, and molecular weight (MW) is indicated on the right. (B) Analysis of SR-BI expression on freshly isolated PTH by flow cytometry is shown. Following fixation and permeabilization, cells were incubated with rabbit anti-SR-BI polyclonal antibody (NB 400-104 at a 1:500 dilution) and subsequently stained with FITC-conjugated goat anti-rabbit IgG. Negative control (NC) represents PTH incubated with control antibody. x and y axes show mean fluorescence intensity and relative number of stained cells, respectively.

FIG. 3.

E2 binding to CHO cells transfected with mouse, human, and tupaia SR-BI. CHO cells were transfected with expression constructs containing the cDNA for mouse (pcDNA3/mSR-BI), human (pcDNA3/hSR-BI), tupaia SR-BI (pcDNA3/tSR-BI), or control vector (pcDNA3) as described previously (36). (A) FACS analysis of anti-SR-BI binding (using polyclonal rabbit anti-SR-BI interacting with both human and mouse SR-BI in permeabilized CHO cells [36]) in transfected CHO cells indicates that approximately 30% of cells transfected express the receptor on the cell surface. (B) FACS analysis of E2 binding to transfected CHO cells shows comparable E2 binding to human and tupaia SR-BI but not to mouse SR-BI.

FIG. 4.

(A) Dose-dependent and saturable binding of E2 to PTH. Hepatocytes were incubated with His-tagged E2 at the concentrations indicated. Cellular E2 binding (corresponding to net mean fluorescence intensity [ΔMFI]) was determined by FACS analysis using an anti-His-biotinylated mouse antibody and streptavidin-R-PE as described in Materials and Methods. (B) E2 binding in the presence of anti-tupaia SR-BI antibody. PTH were incubated with anti-tupaia SR-BI antiserum (black shadowed graph) or preimmune serum (grey shadowed graph) 1 h prior to the addition of recombinant E2 (E2 concentration of ∼4 μg/ml, antiserum dilution of 1:10). Negative control (NC) representing PTH incubated with preimmune serum (1:10 dilution), anti-His-biotinylated mouse antibody, and streptavidin-R-PE in the absence of E2 protein is shown. Cellular E2 binding was analyzed by FACS analysis as described above. (C) Dose-dependent inhibition of E2 binding to PTH by anti-tupaia SR-BI antiserum. PTH were preincubated with different dilutions of anti-tupaia SR-BI (squares) or preimmune serum (circles). After washing with PBS, PTH were incubated with recombinant E2 (E2 concentration of ∼1.5 μg/ml) and cellular E2 binding was analyzed by FACS analysis as described for panel A. Data are shown as percent binding compared to binding of E2 in the presence of PBS (at 100%) of a representative experiment. (D) Inhibition of cellular E2 binding by anti-tupaia SR-BI and anti-human SR-BI antibodies. PTH were preincubated with anti-tupaia SR-BI, anti-human SR-BI, or control antibody (preimmune serum, all diluted 1:10 in PBS) and subsequently analyzed for E2 binding (E2 concentration ≈ 1.5 μg/ml) as described for panel A. Data are shown as percent binding (mean ± standard deviation of a representative experiment performed in triplicate) in the presence of antibody compared to binding of E2 in the presence of PBS (at 100%).

FIG.5.

Specific and dose-dependent binding of anti-tupaia and human SR-BI antibodies to tupaia SR-BI. (A) Specific binding of rabbit anti-SR-BI (NB 400-104, left panel), mouse anti-tupaia SR-BI (middle panel), and mouse anti-human SR-BI (right panel) to tupaia SR-BI expressed in CHO cells. CHO cells were transfected with expression constructs containing control vector (pcDNA3) or the cDNA for tupaia SR-BI (pcDNA3/tSR-BI) as described previously (36). Flow cytometry of transfected cells incubated with anti-SR-BI and R-PE or FITC-conjugated secondary antibodies demonstrated specific interaction of anti-SR-BI antibodies with tupaia SR-BI (bottom panels). In contrast, no interaction was present in CHO cells transfected with control vector and incubated with anti-SR-BI antibodies (upper panels). Numbers inside the panel represent the percentage of positively stained cells in relationship to the total number of cells. For staining with anti-SR-BI NB 400-104, cells were permeabilized prior to incubation with antibody. (B) Dose-dependent interaction of anti-SR-BI with tupaia SR-BI expressed in CHO cells. CHO cells were transfected with expression constructs containing control vector (pcDNA3) or cDNA for tupaia SR-BI (pcDNA3/tSR-BI) as described for panel A. Cells were then incubated with anti-tupaia SR-BI in increasing dilutions, and anti-SR-BI binding was determined on nonpermeabilized cells as shown for panel A. The percentages of SR-BI-positive cells specifically recognized by anti-tupaia SR-BI were determined by subtracting the number of positive cells in pcDNA3-transfected cells stained with anti-SR-BI (negative control) from the number of positive cells in pcDNA3/tSR-BI-transfected cells stained with anti-SR-BI. Histograms (left panel) and percentages of cells (right panel) recognized by anti-tupaia SR-BI are shown in relationship to antibody dose. (C) Detection of cell surface SR-BI on PTH by using mouse anti-tupaia and human SR-BI antibodies. Nonpermeabilized PTH were incubated with mouse anti-tupaia (left panel) and human SR-BI (right panel) sera (1:500 dilution) or mouse preimmune serum (1:500 dilution) and subsequently stained with R-PE-conjugated goat anti-mouse IgG. x and y axes show mean fluorescence intensity and relative number of stained cells, respectively.

FIG. 6.

(A) Dose-dependent and saturable binding of HCV-LPs to PTH and human HepG2 cells. Cells were incubated with increasing concentrations of HCV-LPs. After washing with PBS, cellular binding of HCV-LPs was analyzed by flow cytometry using a mouse monoclonal anti-E2 antibody and PE-conjugated anti-mouse IgG. On the y axis, net mean fluorescence intensity (ΔMFI) values for each HCV-LP E2 concentration were calculated by subtracting the MFI of the negative control (control insect cell preparation) with anti-E2 and PE-conjugated anti-mouse IgG antibodies from that obtained with the respective HCV-LP E2 concentration (x axis). Squares, binding of HCV-LPs to PTH; triangles, binding of HCV-LPs to HepG2 cells. (B) SR-BI-dependent binding of HCV-LPs to PTH. PTH were preincubated with anti-tupaia SR-BI or control antibody (in preimmune serum at a 1:10 dilution). After washing with PBS, PTH were incubated with HCV-LPs in subsaturating concentrations (HCV-LP E2 concentration of ∼1 μg/ml) and cellular HCV-LP binding was assessed using a chimpanzee monoclonal anti-E2 antibody as described in Materials and Methods.Data are shown as percent binding (mean ± standard deviation [SD] of a representative experiment performed in triplicate) in the presence of antibody compared to binding of HCV-LPs in the presence of PBS (at 100%). (C) SR-BI-dependent binding of HCV-LPs to human HepG2 hepatoma cells. HepG2 cells were incubated with anti-human SR-BI or control antibody (in preimmune serum at a 1:10 dilution) 1 h prior to the addition of HCV-LPs. After washing with PBS, HepG2 cells were incubated with HCV-LPs in subsaturating concentrations (HCV-LP E2 concentration of ∼1 μg/ml) and cellular HCV-LP binding was assessed as described for panel B. Data are shown as percent binding (mean ± SD of a representative experiment performed in triplicate) in the presence of antibody compared to binding of E2 in the presence of PBS (at 100%).

FIG.7.

(A) Sensitivity and specificity of HCV RNA detection from hepatocytes by strand-specific RT-PCR. Different amounts of in vitro-synthesized positive- or negative-strand HCV RNAs (synthesized as described in Materials and Methods) were added to the total cellular RNAs of uninfected PTH, respectively, to study the sensitivity and specificity of HCV RNA detection using strand-specific RT-PCR. Positive- and negative-strand HCV RNAs were then analyzed by strand-specific RT-PCR using rTth polymerase 5′-UTR-specific primers (23, 46). Sensitivity for both strands ranged from 0.1 to 1 fg per assay for the correct RNA template, while for the incorrect template (use synthetic negative-strand HCV RNA as template for the detection of positive-strand HCV RNA or vice versa), at least 0.1 pg of the template was needed to give a false-positive signal. (B) Infection of PTH by plasma-derived HCV. PTH were incubated with HCV RNA-positive plasma (samples M1, M2, G3, and G4, all HCV genotype 1) on day 2 after plating as described in Materials and Methods. HCV infection was determined by strand-specific RT-PCR of cellular positive- and negative-strand HCV RNAs on days 1 and 5 postincubation (p. i.) in PTH as shown for panel A and described in Materials and Methods. (C) HCV infection of PTH in the presence of anti-tupaia SR-BI. PTH were preincubated with anti-tupaia SR-BI (left panel) or preimmune serum (right panel) at a dilution of 1:10 1 h prior to the addition of HCV plasma (samples M2 and G4, both genotype 1). Following the washing of nonbound anti-SR-BI, plasma-derived HCV was added to the cell culture medium. The MOIs (calculated as the number of HCV genomic equivalents present in the inoculum divided by the number of hepatocytes) were 0.05 (3 × 10⁴ genomic equivalents/6 × 10⁵ hepatocytes) and 0.25 (1.5 × 10⁵ genomic equivalents/6 × 10⁵ hepatocytes) for plasma samples M2 and G4, respectively. HCV infection was determined by strand-specific RT-PCR as described for panel A. To exclude that SR-BI molecules were accessible for HCV-E2 at the hepatocyte cell surface via the de novo expression or intracellular pool redistribution of SR-BI during the time of viral infection, infection experiments were repeated in the presence of anti-tupaia SR-BI or preimmune serum before and during the time of infection (lower panel). For these experiments, PTH were preincubated for 1 h with anti-tupaia SR-BI or preimmune serum (both serum dilutions of 1:10) and plasma-derived HCV (samples and MOI as described for panel B) was added to the cell culture medium without the removal of non-cell-bound anti-SR-BI. Mock-infected PTH served as negative controls.