18 Nonbound HCVcc were removed by buy GW-572016 washing of cells with phosphate-buffered saline, and cell bound HCV RNA was then quantified by reverse-transcription polymerase chain reaction.18 HCV cell-to-cell transmission was assessed as described.2, 24 Producer Huh7.5.1 cells were electroporated with Jc1 RNA33 and cultured with naïve target Huh7.5-GFP cells in the presence or absence of anti–SR-BI or control mAbs. An HCV E2-neutralizing antibody (AP33, 25 μg/mL) was added to block cell-free transmission.24 After 24 hours of coculture, cells were fixed with paraformaldehyde, stained with an NS5A-specific antibody (Virostat),

and analyzed via flow cytometry.2, 24 Cell spread was assessed by visualizing Jc1-infected Huh7.5.1 cells by immunoflorescence using anti-NS5A (Virostat) and anti-E2 (CBH23) antibodies as described.2 HDL was labeled using Amersham Cy5 Mono-Reactive Dye Pack (GE Healthcare). Unbound Cy5 was removed by applying labeled HDL on illustra MicroSpin G-25 Columns (GE Healthcare). Blocking of Cy5-HDL binding with indicated reagents was performed for 1 hour at room temperature prior to Cy5-HDL binding for 1 hour at 4°C on 106 target cells. Selective HDL-CE uptake and lipid efflux assays were Erlotinib performed as described.23, 34 HDL-CE uptake was

assessed in the presence or absence of anti–SR-BI mAbs (20 μg/mL) and 3H-CE-labeled HDL (60 μg protein) for 5 hours at 37°C. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and is denoted in

μg HDL-CE/μg cell protein. For lipid efflux assay, Huh7 cells were labeled with 3H-cholesterol (1 μCi/mL) and incubated at 37°C for 48 hours as described.23, 35 Cells were incubated with anti–SR-BI mAbs (20 μg/mL) for 1 hours prior to incubation with unlabeled HDL for 4 hours. Fractional cholesterol efflux was calculated as the amount of label obtained in the medium divided by the total in each well (radioactivity in the medium + radioactivity in the cells) regained after lipid extraction from cells. Unless otherwise stated, MCE data are presented as the means ± SD of three independent experiments. Statistical analyses were performed using a Student t test and/or Mann-Whitney test; P < 0.01 was considered statistically significant. To further explore the role of HCV–SR-BI interaction during HCV infection, we generated five rat and three mouse monoclonal antibodies (mAbs) directed against the human SR-BI (hSR-BI) ectodomain (Table 1). These antibodies bound to endogenous SR-BI on human hepatoma Huh7.5.1 cells and primary human hepatocytes but did not bind to mouse SR-BI (mSR-BI) expressed on rat BRL cells (Fig. 1A,B and Supporting Fig. 1). Three rat mAbs (QQ-4A3-A1, QQ-2A10-A5, and QQ-4G9-A6) and one mouse mAb (NK-8H5-E3) significantly (P < 0.01) inhibited HCVcc infection in a dose-dependent manner with 50% inhibitory concentrations (IC50) between 0.