Differences in Western Blotting and human miRNA analyses between immunoprecipitated HBsAg particles and control immunoprecipitations.

<p>Left-most: Western blotting analysis of protein lysates from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs) for detection of Ago2 protein. IP+ lanes contain pooled protein lysates from samples # 1–13 and single lysates from samples # 1 and 9 respectively. Ctrl-IPs lanes contain protein lysates from immunoprecipitates obtained from HBsAg positive sera with mouse monoclonal anti-human c-myc antibody (unrelated-IP+) and HBsAg negative sera using anti-HBs monoclonal antibody (HBs-IP−). HEK is the protein lysate from HEK cells used as positive control for Ago2 protein. The figure is representative of 3 independent experiments. Right-most: HCL is unsupervised hierarchical cluster analysis of detected miRNAs (DC_T values) in both HBs-immunoprecipitated fractions from HBsAg positive immunoprecipitates (HBs-IP+) and control HBsAg negative immunoprecipitates (ctrl-IPs). GDM is supervised gene distance matrix correlating DCt values of HBs-IP+ vs ctrl-IP samples.</p

Identification of human miRNAs associated with serum HBsAg.

<p>(a) The circulating HBsAg-miRNA signature: average RQs were obtained from the comparative DDC_T analysis. Values are reported in a bar plot as a logarithmic scale base 10 along with SD. (b) Differentially detected miRNAs between HBsAg positive immunoprecipitates (HBs-IP+) samples (left-most, n = 11; right-most, n = 4) and the group of control HBsAg negative immunoprecipiates (ctrl-IPs) (n = 4) were selected by Mann-Whitney test on –DCt values (left-most, p<0.1; right-most, p<0.05), and an unsupervised hierarchical cluster analysis was finally performed. Venn diagrams indicate the comparison among the pool of HBsAg-associated miRNAs obtained from the comparative DDC_T analysis of panel a (blu circle) and the HBsAg-associated miRNAs obtained from the Mann-Whitney tests (red circle).</p

Demographic and Virologic Characteristics of Individuals and Sera.

<p>Abbreviations: VP = viral particles, virion; SVP = subviral HBsAg particles; IU = International Units; ng = nanograms; ALT = serum alanine amino trasferase.</p><p>Quantitative values for VP and SVP were obtained as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Gerlich1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Desire1" target="_blank">[15]</a>: 1 IU of HBsAg corresponds to 1,1E+06 IU HBV DNA and1 ng of HBsAg corresponds to 2,08E+08 SVP or 5,0E+07 VP.</p><p>HBV infection and disease phases were characterized as previuosly reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone.0031952-Brunetto1" target="_blank">[8]</a>: IC = Inactive HBsAg Carriers with serum HBV-DNA persistently below 2000 IU and without liver disease; AC1 = Active HBsAg Carriers with serum HBV-DNA fluctuating below 20.000 IU with normal liver histology; AC2 = Active HBsAg carriers with serum HBV-DNA fluctuating above 20.000 IU with chronic active hepatitis at histology, patients with chronic hepatitis B (CHB).</p

Canonical pathways enriched by predicted targets of HBsAg-associated miRNAs.

<p>Target genes of HBsAg-associated miRNAs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3a</a>) were predicted (TargetScan) and ranked. Functional analysis (IPA) was then applied on predicted target genes, and canonical pathways were finally ranked according to their significance in a Fisher exact test (p-value≤0.05, horizontal threshold line in the plot). Left axis: –log(p-values) as histogram boxes; the asterisk (*) marks the three pathways that resulted as significantly enriched by the analyzed gene-list; right axis: for each pathway, black dots indicate the ratio of pathway-defining genes, which is calculated as the number of involved target genes over the total number of genes in that pathway reference dataset.</p

HBs-immunoprecipitation led to a significant change of detection of some human miRNAs in examined sera.

<p>Heatmap of differentially detected miRNAs in whole HBsAg sera (S+) and HBsAg positive flowthroughs after immunoprecipitation (HBs-F+) was obtained by Mann-Whitney test (p<0.05) followed by hierarchical clustering (-DC_T are represented). Venn diagram: the 157 differentially abundant serum miRNAs between whole HBsAg positive sera (S+) and HBsAg positive flowthroughs (HBs-F+) were compared to miRNAs of clusters 1-to-5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031952#pone-0031952-g003" target="_blank">Figure 3b</a> (right panel).</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

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

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