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. 2015 Feb 4;89(8):4387–4404. doi: 10.1128/JVI.02705-14

FIG 2.

FIG 2

AAK1 and GAK regulate HCV entry at a postbinding step. (A) Cells transfected with siRNAs targeting AAK1, GAK, and SDC1 or an NT sequence as well as NT cells pretreated with heparinase I were infected with HCVcc at 4°C for 2 h. This was followed by removal of unbound virus, RNA extraction, and quantification of cell-bound HCV RNA by qRT-PCR as a measurement of HCV attachment. Shown are SDC1 protein levels in the indicated cells and HCV RNA levels relative to the NT control level. (B) Purification of highly infectious Dil-HCV particles. Concentrated HCV was labeled with Dil, layered on a 10 to 80% sucrose gradient, and purified by ultracentrifugation. The top of the gradient is fraction 1. Infectious HCV (solid line, left y axis) was quantified by focus formation (FF) assays, and HCV RNA copy numbers (dashed line, right y axis) were determined by RT-PCR. Dil-HCV was isolated from gradient fraction 3, which harbored the peak of infectivity and had a density of 1.06g/ml. (C to F) Quantitative immunofluorescence analysis of Dil-HCV internalization. Huh-7.5 cells transfected with an NT (D), AAK1 (E), or GAK (F) siRNA and expressing the early endosomal marker RAB5-GFP were infected with Dil-HCV for 1 h on ice and shifted to 37°C. Cells were fixed 1 h postinfection. Images were acquired on a DeltaVision OMX Blaze system equipped with three emCCDs. Colocalization of Dil-HCV and RAB5 was quantified by Pearson's colocalization coefficient (C). Each symbol represents an individual Dil-HCV particle; horizontal lines indicate the means ± standard deviations (error bars) (n = 121, 78, and 84 Dil-HCV particles in the NT and AAK1- and GAK-depleted cells, respectively). Data are from two independent experiments. Two representatives of Dil-HCV (red), RAB5 (green), and merged images at a magnification of ×100 are shown for each group (D to F). The small frames at the bottom correspond to boxed areas and areas with arrows. Scale bar, 10 μm. Arrows indicate Dil-HCV-RAB5 colocalization. Boxed areas indicate Dil-HCV particles that are not colocalizing with RAB5.