PI4KA colocalizes with HCV membranous webs and its silencing leads to aberrant web morphology.

<p>(<b>A</b>) Silencing of PI4KA leads to the disappearance of NS5A-GFP expression in HCV replicon cells. Huh7.5.1 cells expressing a subgenomic replicon were transduced with a nontargeting (left) or PI4KA-targeting (right) lentiviral shRNA vector for 5 days. HCV membranous web distribution was visualized using GFP-tagged NS5A. Bar, 20 µm. (<b>B</b>) Silencing of PI4KA leads to accumulation of membrane “clusters” in a nonreplicative model of web assembly. Huh7/T7 cells were transduced with a nontargeting (upper row) or PI4KA-targeting (lower rows) lentiviral shRNA vector and then transfected with pTM1(NS3-5B/GFP) to express the subgenomic NS3-5B polyprotein (with GFP-tagged NS5A). NS3 and NS4B were visualized by immunofluorescence staining and nuclei were counterstained with DAPI. Bars, 10 µm. (<b>C</b>) Huh7/T7 cells transduced with a nontargeting (NT) or PI4KA-targeting shRNA vector were transfected with pTM1(NS3-5B/GFP). Cells were then counted to determine the fraction with the membrane “cluster” phenotype. At least 100 cells were counted for each condition in each of three independent experiments. Values shown are means ± SD. (<b>D</b>) Colocalization of endogenous PI4KA immunofluorescence staining with NS5A in Huh7.5.1 cells expressing a subgenomic replicon (upper panels) or in Huh7/T7 cells transfected with pTM1(NS3-5B). Nuclei were counterstained with DAPI. Bars, 10 µm. (<b>E</b>) PI4KA requires kinase activity to support HCV replication. OR6 replicon cells were transduced with MMLV retroviral vectors encoding GFP, PI4KA, or a PI4KA D1899A mutant lacking kinase activity. The cells were then transduced 24 h later with lentiviral vectors encoding a nontargeting shRNA (black bars) or a shRNA targeting the PI4KA 3′UTR (white bars) to silence endogenous PI4KA. Cells were assayed 96 h after lentiviral transduction (upper panel). Values were obtained from quadruplicate wells in two independent experiments and are plotted as means ± SD. Cells in duplicate wells were lysed for immunoblotting for PI4KA, anti-FLAG, and beta-actin (lower panel) to confirm PI4KA silencing and PI4KA construct expression. PI4KA band intensities were quantitated by densitometric analysis using NIH ImageJ software and normalized to beta-actin.