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

Time until E_max = 50% for lipid peroxidation sensitive and resistant HCV clones, measured by GLuc assay.

<p>Time until E_max = 50% for lipid peroxidation sensitive and resistant HCV clones, measured by GLuc assay.</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

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

Kinetics of HCV inhibition in infected cell culture following addition of an NS5A inhibitor, monitored using different GLuc reporter viruses.

<p>(A) Inhibition of the gt1a LPO^S virus H77S.3/GLuc2A by elbasvir. (B) Inhibition of the gt2a LPO^R virus JFH-1/QL/GLuc2A by elbasvir. (C) Maximum % inhibition (E_max) of GLuc activity at different time points after addition of elbasvir to Huh7.5 cells infected with different cell culture infectious HCV genomes. LPO^S viruses H77S.3/GLuc2A (gt1a) and N.2/GLuc2A (gt1b) are plotted in red. LPO^R viruses H77D/GLuc2A (gt1a), JFH-1/QL/GLuc2A (gt2a) or HJ3-5/GLuc2A (gt1a/2a chimera). (D) Inhibition of the gt1a LPO^R virus H77D-GLuc2A by elbasvir. (E) Fitness of different virus genomes used in (C) at 72 h post electroporation. Data shown represents GLuc activity relative to GLuc activity at 6 hours post electroporation to normalize for transfection efficiency. The replication incompetent reporter viruses H77S/AAG/GLuc2A and JFH-1/GND/GLuc2A contain point mutations in the NS5B polymerase and are included as mock-transfection controls.</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

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

Differences in kinetics of virus inhibition in H77S.3- vs H77D–infected cells following addition of an NS5A inhibitor are not due to differences in NS5A sequence.

<p>(A) Diagram of the H77S.3 genome showing positions of the 12 amino acids that differ between H77S.3 and H77D. The two amino acid changes in the NS5A coding region (I2204S and D2416G) are highlighted. (B) Maximum % inhibition (E_max) at different time points after addition of elbasvir to Huh7.5 cells infected with either H77S.3/GLuc2A, H77D/GLuc2A or H77S.3/GLuc2A carrying either the I2204S mutation alone (H77S.3/IS) or in combination with D2416G (H77S.3/IS/DG). (C) Fitness of different virus genomes used in (B) at 72 h post electroporation. Data shown represents GLuc activity relative to GLuc activity at 6 h post electroporation to normalize for transfection efficiency. The replication incompetent reporter virus H77S/AAG/GLuc2A contains point mutations in the NS5B polymerase and serves as a mock-transfection control.</p