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

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

Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact.

<p>(A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).</p

Identification of ABHD5 as a new host factor for HCV production.

<p>(<b>a, b</b>) A rational siRNA screen was designed to identify host factors involved in the lipid metabolism and participating in the HCV replication cycle (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005568#ppat.1005568.s002" target="_blank">S2 Fig</a>) with readouts for HCV entry and replication (<b>a</b>) or assembly and release (<b>b</b>). For each graph, the p-value is plotted against the median score. A maximal p-value of 0.05 together with a mean score superior to 2 (blue dots, antiviral factors) or inferior to -2 (green dots, proviral factors) was considered highly significant. CD81, PI4KA and APOE controls are shown in red in the relevant graph and the non-targeting negative control siRNAs in grey. ABHD5 is depicted with a diamond. Yellow dotted lines indicate our statistical thresholds. (<b>c, d</b>) ABHD5-specific siRNAs used in the initial screen as a pool (panels a and b) were transfected individually into HCV RNA-transfected cells. Their specific effect on HCV RNA replication (panel <b>c</b>, corrected for cell viability effects) and progeny virion production (panel <b>d,</b> corrected for HCV RNA replication effects) is depicted after normalisation to the average value of two non-targeting siRNAs. Note that statistics were performed at the gene level. (<b>e- g</b>) Effect of ABHD5-specific shRNAs on ABHD5 gene expression (<b>e</b>), HCV entry and replication (<b>f</b>) and HCV assembly and release (<b>g</b>). (<b>e</b>) ABHD5 mRNA levels were quantified by qRT-PCR at the time of virus harvest. (<b>f</b>) HCV entry and replication were determined by the RLuc activity in the producer cell lysates at the same time point and corrected for the effects on cell viability. (<b>g</b>) The efficiency of HCV production was evaluated by the RLuc activity in target cells infected with the supernatant of shRNA-transduced and JcR-2a-infected producer cells, and corrected for the shRNA effects on HCV entry and replication.</p

HCV-replicating cells induce IFN-β induction <i>in vivo</i>.

<p>(A) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ mock transfected (left flank) or HCV subgenomic RNA transfected (SGR2; right flank) Huh7.5 cells. At the indicated time points post s.c. injection, luciferin was injected i.v. and luciferase activity was measured using the IVIS Spectrum <i>in vivo</i> imaging system. (B) Quantification of luciferase activity. Each symbol represents a region of interest (ROI) analysis of an individual animal (n = 6). (C) IFN-β^+/Δβluc mice were s.c. injected with 5x10⁶ pUCΔGDD (ΔGDD) transfected or HCV SGR RNA transfected Huh7.5 cells treated with or without the HCV protease inhibitor telaprevir. At the indicated time points post s.c. injection, luciferin was injected i.v., the luciferase activity measured using the IVIS Spectrum <i>in vivo</i> imaging system and the signal quantified. Each symbol represents an individual animal (n = 3–4). (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; 2-way ANOVA, means + SD; n.s. not significant).</p