Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor

Abstract

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ∼170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture-derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol-dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

Conflict of interest statement

Conflict of interest

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature.

Figures

Figure 1

NPC1L1 plays a role in HCVcc infection. (a) NPC1L1 topology. (b) Immunoblot of NPC1L1, HCV NS3, and β-actin in Huh7 cells mock-infected or infected with HCVcc at an MOI of 3.0 FFU cell−1 over the course of 12 d. (c–e) Huh7 cells were mock-transfected or transfected with irrelevant control (siCon), SR-BI-specific, CD81-specfic, or NPC1L1-specific siRNAs and subsequently infected with HCVcc at an MOI of 0.05 FFU cell−1 at indicated times post-transfection. (c) Forty-eight h p.i. HCV RNA was quantified by RTqPCR and data normalized to GAPDH. Results are graphed as a percentage of infection achieved in siCon-transfected cultures. (d) NPC1L1 transcript levels were quantified by RTqPCR, normalized to GAPDH and are graphed as a percentage of the maximum number of copies determined in siCon-transfected cultures at each time point examined. (e) Immunoblot of NPC1L1 and β-actin protein expression in siCon-transfected (–) and siNPC1L1-transfected cultures (+). (f,g) Huh7 cells were treated with 36 µg ml−1 of indicated antibodies for 1 h prior to and during HCVcc infection at an MOI of 0.05 FFU cell−1. HCV RNA levels were determined by RTqPCR analysis 24 (f) or 48 (f and g) h p.i. Data were normalized to GAPDH levels and results are graphed as a percentage of infection achieved in respective IgG control-treated cultures. In all cases, significant differences relative to controls (one-way ANOVA and Tukey’s post hoc t test) are denoted as * P < 0.05 or ** P <0.01. All results are graphed as means ± SD for triplicate samples. The data presented are representative of three independent experiments.

Figure 2

Ezetimibe-mediated inhibition of NPC1L1 reduces HCV entry at a post-binding pre-fusion step. (a–c) Huh7 cells were vehicle-treated or treated with increasing concentrations of ezetimibe (a) for 6 h prior to infection and then removed (PRE), (b) for 12 h coincident with viral inoculation and then removed (CO), or (c) following viral inoculation (POST) with HCVcc at an MOI of 1.0 or 0.1 FFU cell−1. HCV foci were quantified 72 h p.i. and are expressed as a percentage of the foci obtained in vehicle-treated (0 µM) cultures ± SD (n = 3). (d–g) Huh7 cells were treated with vehicle or indicated concentrations of ezetimibe beginning 1 h prior to and during infection with (d) HCVcc containing the structural region of the indicated genotypes or (e–g) HCVcc JFH-1 at an MOI of 0.1 FFU cell−1. HCV RNA was quantified by RTqPCR at the indicated times p.i. and data normalized to GAPDH. Results are graphed as a percentage of infection in vehicle-treated cultures or as mean HCV RNA copies/μg total cellular RNA ± SD (n = 3). Assay background (BG) = HCV RNA level detected in uninfected samples. (f,g) Indirect immunofluorescence analysis of HCV NS5A in (f) vehicle-treated and (g) ezetimibe-treated cultures 24 h p.i. Scale bar = 20 µm. (h) Synchronized infections in Huh7 cells were treated with vehicle or ezetimibe (30 µM) at the indicated times. Thirty hours p.i. RNA was harvested. HCV RNA was quantified by RTqPCR, normalized to GAPDH and displayed as mean HCV RNA copies/μg total cellular RNA ± SD (n = 3). Significant reduction in HCV relative to vehicle-treated cultures (one-way ANOVA and Tukey’s post hoc t test) is denoted as * P < 0.05 or ** P <0.01. (i,j) Huh7 cells were treated with vehicle (UT), NH4Cl (10 mM), ezetimibe (30 µM), IgG control antibody (36 µg ml−1), anti-CD81 antibody (36 µg ml−1), or anti-NPC1L1 LEL1 antibody (36 µg ml−1) beginning 1 h prior to inoculation with HCVccDiD (MOI of 5.0 FFU cell−1). HCV fusion was measured by DiD dequenching every 6 min. Results are graphed as a percentage of maximum background-corrected relative fluorescence units (RFU) achieved in vehicle-treated or IgG control-treated cultures. All data presented are representative of three independent experiments.

Figure 3

NPC1L1-mediated HCV cell entry is cholesterol-dependent. (a) Cholesterol content of HCVcc JFH-1, HCVcc JFH-1G451R and JFHpp determined by a fluorometric cholesterol quantification assay, after HiTrap™ Heparin HP affinity column purification. Cholesterol content is graphed as cholesterol (nM) per 1×106 genome copies determined by RTqPCR. (b,c) Huh7 cells were mock-transfected or transfected with indicated siRNA, knockdown was confirmed by RTqPCR (data not shown) and cultures were infected with (b) equal titers of JFHpp or VSVGpp or (c) HCVcc JFH-1 or HCVcc JFH-1G451R at an MOI of 0.05 FFU cell−1. JFHpp and VSVGpp infection was determined 72 h p.i and is expressed as relative light units (RLU) ± SD (n = 3). HCV RNA levels were determined by RTqPCR 48 h p.i., normalized to GAPDH and are graphed as a percentage of maximum determined in siCon-transfected cultures. (d,e) Huh7 cells were treated with vehicle or increasing concentrations of ezetimibe beginning 1 h prior to inoculation with (d) equal titers of JFHpp or VSVGpp or (e) HCVcc JFH-1 or HCVcc JFH-1G451R at an MOI of 0.05 FFU cell−1. JFHpp and VSVGpp infection was determined 72 h p.i and is expressed as RLU ± SD (n = 3). HCV RNA was quantified by RTqPCR, normalized to GAPDH and is displayed as mean HCV RNA copies/μg total cellular RNA ± SD. Significant reductions in RNA or RLU values relative to siCon-transfected or vehicle-treated cultures (one-way ANOVA and Tukey’s post hoc t test) are denoted as * P < 0.05 or ** P <0.01.

Figure 4

Ezetimibe delays the establishment of HCV infection in hepatic xenorepopulated mice. (a) Schematic diagram of experiment in which uPA-SCID mice transplanted with human hepatocytes were pre-treated with diluent alone (n = 4 – 5) or ezetimibe (n = 7, 10 mg kg−1 day−1), via oral gavage, starting 2 weeks, 1 week or 2 d prior to infection (indicated by grey bars). The mice were intravenously inoculated on d 0 with HCV human serum containing 1.0×105 genome copies of HCV genotype 1b (indicated by arrow) and treatments were continued as indicated (black bars). (b,c) Serum samples were obtained weekly for three weeks post-infection for HCV RNA determination. Graphed are HCV RNA levels (genome copies ml−1 of serum) one week post-infection from mice pre-treated for (b) two weeks or (c) one week. The lower limit of HCV RNA detection is equal to 102 genomic copies ml−1 of serum. A 2-tailed Fisher’s exact test was performed to compare categorical variables. In all cases P < 0.05 was used to reject the null hypothesis that the distribution of HCV-positive/HCV-negative mice between ezetimibe-treated and nine diluent-treated mice at specific weeks post-infection were the same.