New PI4P pools emerge as a consequence of HCV RNA replication.
A: Scheme of the transcomplementation experiments shown in panel B–D: Huh7-Lunet cells constitutively expressing HCV NS3 to NS5A (I) or NS5A (II), respectively, were transfected with reporter replicons containing luciferase and eGFP genes as indicated to analyze for conditions rescuing RNA replication. B: Wiltype (wt-eGFP), repHIT (repHIT-eGFP) and ΔGDD reporter replicons of genotype 2a (JFH-1) were transfected into Huh7-Lunet cell lines constitutively expressing NS3-5A or NS5A of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates at 24 h, 48 h and 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of a representative of two experiments performed in duplicates. C. Immunofluorescence analysis of the experiment shown in panel B at 48 h post electroporation. GFP (green) or PI4P (red), respectively, was detected with specific antibodies and DAPI was used to stain nuclei (blue). D. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel C. Data represent mean arbitrary units (AU) +/− SD of 35 GFP positive cells analyzed per condition. In case of ΔGDD, cells were randomly chosen due to the lack of GFP signals. Significance was calculated by a paired students t-test. ***, p<0.001.