Inhibitory dose-response curve of AL-9 for purified PI4KIIIα and PI4KIIIβ.

<p>The enzymes were preincubated for 10 min with the indicated concentrations of AL-9 or DMSO and the reaction was started by addition of 100 µM ATP and 150 µM PI∶PS substrate as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Activity, measured as conversion of ATP to ADP, is expressed as percent of the DMSO control. Shown is a representative experiment of three independent experiments performed in duplicate. IC₅₀ and SD of PI4KIIIα and PI4KIIIβ are indicated.</p

AL-9 inhibits PI4KIIIα in Huh7.5 cells.

<p>(A) Confocal microscopy images of Huh7.5 cells treated for 2 hours with DMSO (left column) or with 1, 2, 4 or 8 µM of AL-9 (columns 2 to 5). PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color (zoomed sections are indicated by a yellow square). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. **, p<0.01; ***, p<0.001.</p

Inhibition of PI4KIIIα by AL-9 induces the formation of large NS5A clusters.

<p>Huh7-Lunet/T7 cells were transiently transfected with the plasmid pTM-NS3-5B which expresses the HCV nonstructural proteins under the control of the T7 RNA polymerase promoter. Cells were treated with DMSO (upper panels) or 8 µM AL-9 (lower panels) for 2, 8 or 16 hours and were then stained for NS5A (red) and PI4P (green) using the Golgi staining protocol as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Nuclear DNA was stained with the Hoechst dye (blue). Zoomed sections are indicated by a yellow square. Long incubation with AL-9 (8–16 hours) results in increased NS5A clustering and concomitantly a decrease of PI4P in the internal membranes.</p

Reversibility of HCV-induced changes in PI4P subcellular distribution.

<p>JFH-A4 cells were incubated for 14 days with the HCV RdRP inhibitor HCV-796 (2 µM) or the HCV NS3/4A protease inhibitor MK-5172 (0.2 µM). Cure from HCV was controlled by detection of NS5A with a specific NS5A antibody (red, right column). As control, untreated Huh7.5 cells or JFH-A4 cells were used. Cells were fixed and PI4P (green) was detected in the internal membranes (IM, left column) or in the plasma membrane (PM, central column). For internal membrane staining giantin (red) was used as a specific marker for Golgi membranes. Nuclei were stained by the Hoechst dye (blue).</p

HCV impacts subcellular PI4P distribution.

<p>(A) Huh7.5 cells, JFH-A4 and Con1-SR cells were analyzed by confocal microscopy for the presence of PI4P (green) in the plasma membranes (PM, upper panel) or in the intracellular membrane (IM, lower panel) using the protocols described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Nuclei were stained by the Hoechst dye (blue). For internal membrane staining, giantin (red) was used as a specific marker for Golgi membranes. (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (Huh7.5 cells) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

RNA interference analysis of PI4P production in Huh7.5 cells.

<p>Huh7.5 cells were treated with irrelevant (mock) siRNA or siRNA targeting PI4KIIIα or PI4KIIIβ as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. The data were collected three days after initial siRNA transfection. (A) Confocal microscopy images of Huh7.5 cells treated with PI4KIIIα siRNA, PI4KIIIβ siRNA or mock siRNA. Cells were fixed and stained as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color. (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (mock siRNA) are shown. Four randomly picked fields were analyzed per each condition, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01. (C) Immunoblot analysis of protein expression after RNAi silencing. Lysates prepared from Huh7.5 cells transfected with irrelevant siRNA (mock), PI4KIIIα siRNA (PIKA) or PI4KIIIβ siRNA (PIKB) were analyzed by immunoblotting with PI4KIIIα, PI4KIIIβ or β-actin antibodies as indicated in the figure. Positions of the protein molecular weight markers are shown on the left side.</p

AL-9 inhibits PI4KIIIα in HCV-replicating cells.

<p>(A) JFH-A4 cells were treated with DMSO or AL-9 for 4 hours and internal membranes were stained for PI4P (green) and the Golgi marker giantin (red) using the Golgi staining protocol, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. DMSO or AL-9 concentrations are indicated within the image. Alternatively, cells were stained for NS5A as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a> (indicated as NS5A). Nuclear DNA was stained with Hoechst dye (blue). PI4KIIIα, associated with the HCV-associated membranous web is inhibited by AL-9. The decrease of PI4P is not due to inhibition of the HCV replication indicated by unchanged NS5A expression and localization (lower panel). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. **, p<0.01; ***, p<0.001.</p

Chemical structure of AL-9.

<p>For the synthetic pathway and procedure see Supporting Information.</p

List of EC₅₀ values of AL-9 for different HCV genotypes.

<p>Huh7.5 cells replicating subgenomic replicons of genotype 1b or 2a (Con1-SR and JFH-A4, respectively) or Huh7.5 cells infected with the chimeric virus J6/JFH were treated with AL-9 for three days and intracellular viral RNA was measured by real time PCR. The data are representative of at least three independent experiments, and the standard deviations are shown.</p>*<p>CC₅₀ measured in uninfected Huh7.5 cells.</p

Effect of PIK93 on Golgi or plasma membrane PI4P in Huh7.5 cells.

<p>(A) Confocal microscopy images of Huh7.5 cells incubated with DMSO (left column), 0.5 µM PIK93 (central column) or with 30 µM PIK93 (right column) for 2 hours prior to fixation and staining as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. PI4P (green) localized to the plasma membrane (PM) was detected using the plasma membrane staining protocol (upper panel) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#ppat.1002576-Hammond1" target="_blank">[37]</a>. Nuclei were stained by the Hoechst dye (blue). PI4P in the intracellular membranes (IM) was revealed using the Golgi staining protocol (lower panel). Together with PI4P, Golgi membranes were stained with the Golgi marker giantin (red). Colocalization of PI4P with Golgi membranes results in yellow color (zoomed sections are indicated by a yellow square). (B) Quantification of PI4P levels by immunofluorescence analysis. Changes in mean fluorescence intensity relative to the control (DMSO) are shown. Four randomly picked fields were analyzed per each condition. Normalization was performed as detailed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002576#s4" target="_blank">Materials and Methods</a>. Data are presented as averages ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p