Impact of mutations within the PI4KIIIα binding region on HCV RNA replication and partial rescue by PI4KIIIα overexpression.
A: Schematic representation of a monocistronic replicon used in this study. 5′ non-translated and 3′ non-translated regions are indicated by secondary structures. Luciferase (Luc) is connected by a cleavable ubiquitin (ubi) linker to the non-structural proteins NS3 to NS5B. C, core. B: Huh7-Lunet cells were transfected with luciferase reporter replicons bearing the indicated triple alanine substitutions. JFH-1 wt replicons (wt, green) and a mutant harboring a deletion within NS5B (ΔGDD, red) served as positive and negative controls, respectively. Replication efficiency is expressed as luciferase activity (RLU) at 24 h (light color), 48 h (medium color) and 72 h (dark color) relative to 4 h after transfection to normalize for transfection efficiency. 24 h values of mutants with delayed kinetics are highlighted by red lines. Error bars indicate mean +/− SD of a representative experiment (n = 3). C: Huh7-Lunet cells were transfected with luciferase reporter replicons as described in B. Total cellular RNA was extracted 4 h or 72 h after transfection. HCV RNA was quantified by quantitative RT-PCR and is shown as HCV copies at 72 h relative to 4 h after transfection. Error bars indicate mean +/− SD of a representative experiment (n = 6). D: Naïve Huh7-Lunet cells (control) or Huh7-Lunet cells stably overexpressing HA-tagged PI4KIIIα (HA-PI4KIIIα) were transfected with luciferase reporter replicons as described in B. Replication efficiency is expressed as luciferase activity (RLU) at 48 h relative to 4 h after transfection. Error bars indicate mean +/− SD of quadruplicate values of two independent experiments. E: Overexpression of HA-PI4KIIIα was confirmed by immunoblot of whole cell lysates of naïve Huh-7 Lunet cells (control) or HA-PI4KIIIα overexpressing cells (HA-PI4KIIIα) using antibodies directed against PI4KIIIα, HA-peptide or Calnexin.