The Role of the Phosphatidylinositol 4-Kinase PI4KA in Hepatitis C Virus-Induced Host Membrane Rearrangement
Figure 6
HCV-induced membrane clustering in PI4KA-silenced cells requires NS3-4A protease activity but does not require host secretory pathway integrity.
(A) Formation of membrane “clusters” in PI4KA-silenced cells expressing HCV proteins requires HCV polyprotein cleavage. Huh7/T7 cells were transduced with a nontargeting (upper panels) or a PI4KA-targeting (lower panels) shRNA vector prior to pTM1(NS3-5B) transfection. Cells were treated with 0.5% DMSO (left panels) or with 10 µM BILN-2061 (right panels) for 24 hours prior to fixation and immunostaining for NS5A. Nuclei were counterstained with DAPI. Bar, 10 µm. (B) Formation of membrane “clusters” in PI4KA-silenced cells expressing HCV proteins does not require integrity of the host secretory pathway. Huh7/T7 cells were transduced with a nontargeting (upper panels) or a PI4KA-targeting (lower panels) shRNA vector prior to pTM1(NS3-5B) transfection. Upon transfection, cells were treated with 0.1% ethanol or with 100 ng/mL BFA for 24 hours prior to fixation and immunostaining for NS5A and beta-COP (to demonstrate COPI coatomer dispersal by BFA). Nuclei were counterstained with DAPI. Bar, 10 µm. (C) Cells from the experiments shown in panels (A) and (B) were counted to determine the fraction with the membrane “cluster” phenotype. A minimum of 100 cells were counted for each condition in each of two independent experiments. Values shown are means ± SD.