Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants

Abstract

Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.

FIG. 1.

SR-BI is involved in the early phase of HCV entry. HCV infection was measured 72 h postinfection by RT-qPCR. J6/JFH HCVcc (HCV) was added to Huh-7.5 cells and incubated for 90 min at 4°C. Cells were incubated with an irrelevant isotype IgG control, anti-SR-BI MAb (C167), or anti-CD81 at different times with respect to the addition of the virus, as indicated at the bottom. Values represent the averages of quadruplicates and are expressed as percentages of HCV infection relative to the cells treated with the isotype IgG control at each time point.

FIG. 2.

E2 binding to SR-BI molecules from different species. N2A cells were transfected with SR-BI cDNAs cloned from livers of different species that were either permissive (human and tupaia SR-BI molecules) or nonpermissive (mouse and tamarin SR-BI molecules) to HCV infection. sE2 binding (genotype 1a, strain H77) was measured 48 h after transfection by flow cytometry. Background was defined as E2 binding to N2a cells transfected with the empty vector. MFI values, subtracted of background MFI values, were expressed as percentages of sE2 binding relative to sE2 binding measured in cells transfected with human SR-BI.

FIG. 3.

E2 binding to SR-BI mutant receptors. (A) The human SR-BI (hSR-BI) and mouse SR-BI (mSR-BI) proteins are schematically depicted at the top, and the transmembrane (TM) domains are indicated. Swap mutants SW0, SW1, and SW2 were generated by replacing the human SR-BI sequence (black) with the murine counterpart (white), and the numbers corresponding to the positions of the amino acids exchanged are shown. Ten single-point mutants were also designed by alanine scanning and are indicated by lanes and numbers. (B) N2A cells were transfected with SR-BI mutant receptors, and sE2 binding (genotype 1a, strain H77) was measured by flow cytometry. Background was defined as the E2 binding to N2a cells transfected with an empty vector. MFI values, with background MFI subtracted, were expressed as percentages of wild-type human receptor binding after normalization for the protein expression level determined by WB (middle). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 4.

Binding of MAbs C11 and C167 to mutant SR-BI receptors. C11 and C167 binding to N2a cells transfected with human wild-type SR-BI (hSR-BI), SW0, or the E210A mutant was measured 48 h after transfection by flow cytometry. Values are expressed as percentages of MAb binding relative to the binding of the MAb in cells transfected with the wild-type human receptor.

FIG. 5.

HCVcc infection of an SR-BI-negative Huh-7.5 cell line complemented with wild-type and mutant SR-BI receptors. (A) J6/JFH HCVcc (HCV) was added to Huh-7.5/shSR-BIkd cells pretreated or not treated with doxycycline (Dox), and HCV infection was measured 32 h postinfection by RT-qPCR. Data are expressed as the numbers of HCV copies normalized to the number of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) copies per 50 ng of total RNA. The SR-BI protein expression level in Huh-7.5/shSR-BIkd cells with or without doxycycline was determined by WB and is shown (middle). (B) HCVcc infection of Huh-7.5/shSR-BIkd cells treated with doxycycline and transduced with either a control lentivirus (Ctrl lenti), human SR-BI (hSR-BI), mouse SR-BI (mSR-BI), or the E210A or SW0 mutant was measured. Results are expressed as increases in HCV infection compared to cells transduced with the control lentivirus. The relative levels of SR-BI protein expression determined by WB are shown at the bottom.

FIG. 6.

Heterooligomerization of wild-type SR-BI with mutant receptors. HEK-293 cells were cotransfected with constructs encoding c-Myc- and V5-tagged versions of wild-type human SR-BI (hSR-BI/myc + hSR-BI/V5) (lanes 2 and 5), c-Myc-tagged E210A and V5-tagged wild-type human SR-BI (E210A/myc + hSR-BI/V5) (lanes 3 and 6), or empty vector pcDNA3 (lanes 1 and 4). Tagged SR-BI proteins precipitated with either anti-V5 (lanes 1 to 3) or anti-c-Myc (lanes 4 to 6) antibodies were stained with anti-c-Myc antibody (Ab). Molecular mass markers are indicated on the left. Bands migrating with an apparent molecular mass of 85 kDa correspond to tagged SR-BI. The faster-migrating bands correspond to the heavy chain of the immunoprecipitating antibody revealed by peroxidase-labeled anti-mouse IgG.

FIG. 7.

Evaluation of HDL binding to SR-BI mutant receptors and their capacity to mediate cellular FC efflux to HDL. CHO cells were transfected to transiently express either wild-type human SR-BI (hSR-BI) or mouse SR-BI (mSR-BI) receptors or the SW0 or E210A mutant molecules. Control cells were transfected with the empty vector pcDNA3. (A) Twenty-four hours posttransfection, the cells were incubated with biotinylated HDL (20 μg protein/ml for 30 min at 4°C). HDL binding was revealed by the addition of streptavidin-PE and analyzed by flow cytometry. The MFI measured is reported. Values ± standard deviations represent the means of data from five to six independent experiments. MFI values for CHO cells expressing either wild-type or mutant receptors were statistically significantly different from that measured with control cells (P < 0.02). (B) HDL-mediated cellular FC efflux in transfected CHO cells labeled with [³H]cholesterol was determined. Forty-eight hours posttransfection, the cells were incubated in serum-free medium containing HDL (20 μg protein/ml) for 2 h. Cholesterol efflux is expressed as the percentage of total [³H]cholesterol radioactivity released into the medium. Data represent the means (± standard deviations) of data from four independent experiments. FC efflux values determined for CHO cells expressing either wild-type or mutant receptors were statistically significantly different from that measured with control cells (P < 0.0001).