Viral determinants required for BA- mediated stimulation.

<p><b>A:</b> Schematic representation of JFH1 NS3-3′, Con1/ET JFH1/Con1 intergenotypic chimeras. Replication enhancing mutations of Con1/ET are marked as black dots. All viral proteins were JFH1-derived and either the 5′NTR or the terminal end of the 3′ NTR (X-tail) or both were exchanged by those of Con1. <b>B:</b> Lunet G-luc cells were transfected and seeded on a 12-well plate. Indicated bile acids were added after 4 h and luciferase activity was measured 72 h post-electroporation. <b>C:</b> Data were normalized to DMSO control.</p

Genotype and cell type dependent influence of bile acids on HCV RNA-replication.

<p>Lunet G-luc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036029#pone.0036029-Gentzsch1" target="_blank">[19]</a> were transfected with either SG-Con1/ET (<b>A</b>) or SG-JFH1 replicons (<b>B</b>) and seeded on a 96-well plate. After 4 h the medium was changed and different BAs (CA = cholic acid; CDCA = chenodeoxycholic acid; DCA = deoxycholic acid; LCA = lithocholic acid; UDCA = ursodeoxycholic acid) in concentrations ranging from 25 µM–400 µM were added. 48 h later cell viability was measured by gaussia luciferase assays. The symbol † designates concentrations with a cell viability of less than 50% of the DMSO control. 72 h after electroporation cells were lysed and replication was determined using the firefly luciferase assay. Data were normalized to DMSO control. Con1-derived genome segments are depicted in white, JFH1-derived sequences in black, and non-HCV elements are depicted in grey (PI, polio IRES; EI, encephalomyocarditis virus IRES; luc, firefly luciferase). (<b>C</b>) HuH6 cells were transfected with JFH1 replicon RNA and seeded on a 96-well plate. After 4 h, BAs were added and after 72 h cells were lysed and replication was measured using the firefly-luciferase assay. <b>D:</b> Lunet G-luc cells were transfected with Con1/ET (left panel) or Con1/GND (right panel) replicons and seeded on a 12-well plate. Bile acids or DMSO were added 4 h after electroporation. Cells were lysed at given time points; luciferase activity was determined and normalized for the 4 h value. In each case mean values of triplicates and the standard deviation is given.</p

Influence of bile acids on HCV whole life cycle.

<p><b>A:</b> Experimental setup and schematic drawing of the Luc-Jc1 reporter virus genome carrying a firefly luciferase gene and of the gaussia luciferase construct. Lunet G-luc cells were transfected with the chimeric full-length reporter virus genome and seeded on a 96-well plate. 4 h post-electroporation the medium was removed and new medium containing bile acids was added. After 48 h, RNA-replication in the transfected cells was determined by firefly luciferase assays. At the same time, culture fluid of the cells was collected to determine cell viability through G-luc activity and to inoculate naïve Lunet G-luc cells. 48 h later efficiency of virus production and infection was determined by measuring firefly luciferase in the inoculated cells. <b>B:</b> RNA-replication (left) and virus production/infection (right) in the presence or absence of given doses of BAs determined in Lunet G-luc cells. Replication and whole life cycle data were normalized to DMSO control. The symbol † designates concentrations with a cell viability of less than 50% of the DMSO control. <b>C:</b> Analysis of the influence of given BA doses on Luc-Jc1 replication in HuH6 cells (left) and on the infectivity of secreted particles upon inoculation of Lunet G-luc cells. Means values of triplicates and the standard deviation is given.</p

Influence of BAs on HCV particle production and infectivity.

<p>Cells were transfected, seeded and treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036029#pone-0036029-g002" target="_blank">Fig. 2</a>. Release of core protein as a measure of viral particles in the culture fluid was determined by a commercial core-specific ELISA (<b>A</b>). Infectivity of release particles was assessed by inoculation of naïve Lunet G-luc cells (<b>B</b>). Mean values of duplicates and the standard deviation are shown.</p

Detection of HCV replication and virus infectivity in the presence of CDCA using Lunet GFP-NLS-MAVS- reporter cells.

<p>A: Lunet MAVS-GFP reporter cells were transfected with given HCV Con1 full length genomes. Ten days after transfection relocalization of GFP to the nucleus was assessed by fluorescence microscopy. Numbers below the panels depict the percentage of cells displaying GFP in the nucleus +/− standard deviation. B: Lunet cells were transfected with given genomes, co-seeded with naïve Lunet GFP-NLS-MAVS (1∶1) and cocultured in the presence or absence of CDCA cells for four days. Subsequently the number of cells showing a nuclear localized GFP was determined by counting of 50 randomly chosen microscopic fields. The left panel shows an example of two infected cells (white arrow) displaying nuclear localized GFP and a non-infected cell (black arrow) after co-culturing with Lunet cells transfected with Con1/K1846T. The right panel depicts mean values and standard deviations from 2 independent experiments. A significant difference in infection efficiency by addition of 200 µM CDCA is indicated by an asterisk (p = 0.0486).</p

CDCA increases replication of Con1 wild type and cell culture adapted replicons.

<p>Given subgenomic Con1 replicons with or without replication enhancing mutations were transfected into Lunet G-Luc cells. At 4 h post transfection cells culture media were replaced with culture fluid with or without 200 µM CDCA. RNA replication was determined by luciferase assays and is expressed relative to the luciferase activity determined 4 h post transfection.</p

CDCA stimulates replication of full length Con1 genomes with or without adaptive mutations.

<p>Given full length Con1 genomes were transfected into Lunet G-Luc cells. At 4 h post transfection cells culture media were replaced with culture fluid with or without 200 µM CDCA. Intracellular (A) and extracellular (B) levels of HCV core protein reflecting viral translation/RNA replication and secretion of virions, respectively, were determined using a commercial ELISA.</p

The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

<div><p>The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.</p></div

New PI4P pools emerge as a consequence of HCV RNA replication.

<p>A: Scheme of the transcomplementation experiments shown in panel B–D: Huh7-Lunet cells constitutively expressing HCV NS3 to NS5A (I) or NS5A (II), respectively, were transfected with reporter replicons containing luciferase and eGFP genes as indicated to analyze for conditions rescuing RNA replication. B: Wiltype (wt-eGFP), repHIT (repHIT-eGFP) and ΔGDD reporter replicons of genotype 2a (JFH-1) were transfected into Huh7-Lunet cell lines constitutively expressing NS3-5A or NS5A of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates at 24 h, 48 h and 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of a representative of two experiments performed in duplicates. C. Immunofluorescence analysis of the experiment shown in panel B at 48 h post electroporation. GFP (green) or PI4P (red), respectively, was detected with specific antibodies and DAPI was used to stain nuclei (blue). D. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel C. Data represent mean arbitrary units (AU) +/− SD of 35 GFP positive cells analyzed per condition. In case of ΔGDD, cells were randomly chosen due to the lack of GFP signals. Significance was calculated by a paired students t-test. ***, p<0.001.</p

Triple alanine mutants induce ultrastructural changes similar to membranous web structures in PI4KIIIα knockdown cells.

<p>Huh7-Lunet T7 cells (A) or Huh7-Lunet T7 cells with stable PI4KIIIα knockdown (B) were transfected with pTM constructs expressing wt or mutant NS3 to NS5B polyproteins or eGFP. Cells were fixed and prepared for EM analysis 24 h post transfection. Consecutive enlargements of the boxed areas are shown from left to right. Note the heterogeneous membranous web (MW, yellow arrows) in cells expressing the wt polyprotein and the clusters of smaller double-membrane vesicles (DMVs) in shPI4KIIIα cells (B) and in cells expressing mutant polyproteins. Scale bars are given in the lower right of each panel. N, nucleus; LD, lipid droplet; rER, rough endoplasmic reticulum; m, mitochondrium. The number in the upper left of right panels shows the average diameter of 70 double-membranous vesicles (DMV) +/− SD measured for each condition. (C) Average diameter of DMVs detected in cells that had been transfected with constructs and conditions specified on the left and shown in panel A and B. Error bars indicate the mean +/− SD of seventy vesicles. Significance of differences in DMV sizes was measured by a paired t-test and is indicated ***, p<0.001.</p