Fig. 5. PI4P and PI4KIIIα levels in HCV containing cells.

(A) HCV permissive Lunet/CD81 cells were mock treated or infected with Jc1. Cells were fixed at given time points and stained with a NS5A- (red) and a PI4P-specific antibody (green). Nuclear DNA was stained with DAPI (blue). An enlargement of the section indicated by the white square is shown in the crop panel in the bottom. Images were acquired with a confocal microscope. (B) Quantification of PI4P levels by immunofluorescence analysis (green bars) and immunoblot (black bars). For immunofluorescence of mock- or Jc1-transfected cells mean values and standard error of the mean (SEM) of 80 cells per condition are shown. For immunoblot, cells transfected with Jc1 or subgenomic replicons specified in the bottom were lysed after 48 h. Lipids were extracted, spotted onto membranes and PI4P was quantified using the PI4P Mass strip kit (Echelon). Signals were quantified by using the Quantity One software (Bio-Rad). Error bars represent the SEM of three independent experiments. IF, immunofluorescence; IB, immunoblotting; n.t., not tested (C) Lysates used in (B) were prepared from mock- or Jc1-transfected cells at time points post transfection given in the top and analyzed by immunobloting for PI4KIIIα, NS5A and Calnexin (loading control). (D) Immunohistochemistry on snap-frozen liver tissues from a HCV negative person (10H6; Table S4, S5) or a patient with chronic HCV infection (3E6; Table S4, S5). Consecutive liver sections were stained for NS5A, PI4P and core. Note that cells staining positive for viral proteins and PI4P reside in the same region of each section.