Figure 2. HCVcc entry upon ectopic hSR-BI co-expression with other HCV receptors.

(A) Results of HCVcc entry assays, assessed by measuring intracellular HCV RNA level at 4, 12, and 72 hr post-infection, in SK-Hep1 cells ectopically expressing CLDN1 vs. CLDN1 and SR-BI, using cell culture–produced HCVcc in the absence or in the presence of NS3 protease inhibitor and NS5B-dependent RNA synthesis inhibitor (BILN2051 and 2′-C-methyl-adenosine, respectively, kindly provided by FV Chisari). Results are standardized with respect to the HCV RNA level obtained at 72 hr on non-treated SK-Hep1-CLDN1-SR-BI cells (mean±SD; n = 3). Intracellular HCV RNA levels of SK-Hep1-CLDN1-SR-BI was on average 440-fold lower than HCV RNA levels measured in Huh7.5 cell (4.7×10³±2.4×10² genome copies per µg cellular mRNA) using the same HCVcc supernatants. (B) Results of HCVcc entry assays, assessed at 72 hr post-infection, in SK-Hep1 cells ectopically expressing the indicated HCV entry factors, using cell culture–produced HCVcc in the absence (white bars) or presence (black bars) of 0.6 µg/ml cholesterol-HDL. Results are standardized with respect to HCV RNA level obtained in the absence of HDL on wt SR-BI–expressing SK-Hep1-CLDN1 cells (mean±SD; n = 5). Similar experiments in BRL3A-CD81-CLDN1-SR-BI cells allowed detection of HCV RNA at 72 hr, which was specifically increased upon treatment with HDL, and revealed differences upon expression of HCV entry factors (data not shown) consistent with the results obtained with HCVpp. However, kinetic experiments and use of replication inhibitors did not allow firm demonstration of HCV replication in these cells. (C) Dose-response curves for the SR-BI–dependent free cholesterol efflux and for HDL-CE uptake determined in BRL3A-CD81-CLDN1 and SKHep1-CLDN1 cells ectopically expressing, or not, hSR-BI, or in Fu5AH cells expressing high endogenous levels of rat SR-BI. Cholesterol efflux is expressed as the percentage of total labelled ³H-cholesterol released to the medium. Selective HDL-CE uptake is expressed as the percentage of labelled HDL-CE delivered to cells per µg of cell protein. The values represent the means ±SD of three experiments.