FIG 6.

VAPs and Nir2 are required for PI(4)P upregulation in HCV-expressing cells. (A) Control or Nir2 knockout Huh7.5.1 cells were transduced with the indicated lentiviral vectors to express sgRNA-resistant Nir2 or mutants before they were infected with NanoLuc-HCV for 3 days, and luciferase activity was measured. Values are normalized to the level of the control. **, P < 0.001, for results compared to the result with the control. Inset, Huh7.5.1 cell pools as described above were immunoblotted for the indicated proteins. (B) Huh7.5.1/T7 cells stably expressing T7 RNA polymerase were transduced by the indicated sgRNA or shRNA to deplete VAPA/B, Nir2, or OSBP and then transfected with constructs expressing the NS3-5B/GFP polyprotein encoding NS5A-GFP under the control of the T7 promoter. Forty-eight hours later, cells were immunostained for PI(4)P (red) and GFP (green) with DAPI nuclear counterstaining (blue). Arrowheads indicate cells expressing the NS3-5B/GFP polyprotein, while asterisks indicate cells with no NS3-5B/GFP expression. Bar, 20 μm. (C) Quantitation of PI(4)P fluorescence from HCV-expressing cells versus HCV-nonexpressing cells in the same experiment as described for panel B. Each point denotes the fold change of integrated fluorescence signal from a single HCV-expressing cell over the average intensity of HCV-nonexpressing cells, with the mean fold change indicated by the black lines.