Fig. 7. Activation of PI4KIIIα by NS5A.

(A) Analysis of recombinantly expressed and purified HCV nonstructural proteins and PI4KIIIα. NS3 and NS5A were expressed as full-length proteins with N-terminal hexahistidine tag, NS5B was C-terminally tagged and lacked the C-terminal membrane insertion sequence. One µg of each protein was loaded onto a SDS 7 % polyacrylamide gel and proteins were visualized by Coomassie blue staining. (B) Purified PI4KIIIα was incubated with a phosphatidylinositol containing substrate and radiolabeled γ-[³²P] ATP in the presence or absence of the indicated purified viral proteins. Molar ratios of PI4KIIIα and a given viral protein are indicated in the bottom. PI4KIIIα activity was determined by measuring the incorporation of [³²P] into the phosphatidylinositol substrate. As controls, the PI4KIIIα inhibitor Wortmannin (WM; 100 µM) was added to the reaction. Data (mean +/− SD; n = 3 in duplicates) were analyzed using a two-way t-test. P-values below 0.05 or 0.001 are indicated by one or three asterisks, respectively. n.d., not detectable (C) Naïve Huh7-Lunet/T7 cells or cells with stable knock-down of PI4KIIIα expression (sh-PI4KIIIα) or expressing a non-targeting shRNA (sh-NT) were transfected with a NS3 to NS5B polyprotein expression construct under control of the T7 RNA polymerase promoter (pTM-NS3-5B) and analyzed for NS5A (red) and PI4P (green) distribution. A fraction of the cells were treated with 30 µM PIK93 for 16 h. Mock transfected Huh7-Lunet/T7 cells (left column of panels) are shown for reference. Nuclear DNA was stained with DAPI (blue). Images were acquired with a confocal microscope. White arrows point to a regular Golgi-like distribution of PI4P.