Impairment of Type I but Not Type III IFN Signaling by Hepatitis C Virus Infection Influences Antiviral Responses in Primary Human Hepatocytes

<div><p>Peginterferon lambda-1a (Lambda), a type III interferon (IFN), acts through a unique receptor complex with limited cellular expression outside the liver which may result in a differentiated tolerability profile compared to peginterferon alfa (alfa). In Phase 2b clinical studies, Lambda administered in combination with ribavirin (RBV) was efficacious in patients with hepatitis C virus (HCV) infection representing genotypes 1 through 4, and was associated with more rapid declines in HCV RNA compared to alfa plus RBV. To gain insights into potential mechanisms for this finding, we investigated the effects of HCV replication on IFN signaling in primary human hepatocytes (PHH) and in induced hepatocyte-like cells (iHLCs). HCV infection resulted in rapid down-regulation of the type I IFN-α receptor subunit 1 (IFNAR1) transcript in hepatocytes while the transcriptional level of the unique IFN-λ receptor subunit IL28RA was transiently increased. In line with this observation, IFN signaling was selectively impaired in infected cells upon stimulation with alfa but not in response to Lambda. Importantly, in contrast to alfa, Lambda was able to induce IFN-stimulated gene (ISG) expression in HCV-infected hepatocytes, reflecting the onset of innate responses. Moreover, global transcriptome analysis in hepatocytes indicated that Lambda stimulation prolonged the expression of various ISGs that are potentially beneficial to antiviral defense mechanisms. Collectively, these observed effects of HCV infection on IFN receptor expression and signaling within infected hepatocytes provide a possible explanation for the more pronounced early virologic responses observed in patients treated with Lambda compared to alfa.</p></div

Productive HCV replication represses IFNAR1 expression in infected hepatocytes.

<p>iHLCs were infected with HCVcc (MOI of 0.2), GT-1b HCVser or mock infected, and subsequently maintained in culture with medium replacement every 2 days. At 24 h post-infection, the NS3 PI ASV (0.5 μM) or vehicle control dimethyl sulfoxide (DMSO) was added to cell cultures during media replenishment. (A) Persistent HCVcc replication as measured by the detection of the virally-encoded core antigen and expression of type I (IFNAR1, IFNAR2) and type III IFN (IL28RA, IL10RB) co-receptor subunits in cells were monitored by Western immunoblotting at the indicated time points, with β-actin used as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVcc infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of three independent Western immunoblot analyses. (B) Co-localization of IFNAR1 and HCV-core positive iHLCs were assessed by fluorescence microscopy. On Day 6 post-infection, naive and HCVcc-infected iHLCs were fixed, permeabilized and stained with antibodies to IFNAR1 (red) and HCV core antigen (green) as indicated. Nuclei were counterstained with Hoechst dye (blue) as shown in the overlays. (C) iHLC cultures infected in parallel with GT-1b HCVser or HCVcc were harvested at the indicated time points and IFNAR1 copy numbers estimated by quantitative RT-PCR following normalization to cellular GAPDH levels in each sample. Results are expressed as mean ± standard deviations (n = 4). Statistical analysis was performed by Bonferroni’s multiple comparison tests: **, <i>P</i> < 0.05; ***, <i>P</i> < 0.001. (D) Cell lysates were harvested at the indicated time points and protein levels of IFNAR1 and IL28RA were examined by Western immunoblotting. Susceptibility of HCVser to the NS3 PI ASV was determined using an antibody directed against NS3. Arrows indicate the presence of the processed and unprocessed forms of the HCV-encoded NS3/4A protease in infected iHLCs. Detection of β-actin served as loading control. Right panel; Relative intensity of IFNAR1 detected in HCVser infected cells maintained in the presence (open triangle) or absence (open square) of ASV treatment was quantified by densitometry analysis, normalized to β-actin levels in each sample, and expressed as a percentage relative to mock-infected cells. Data are representative of two independent Western immunoblot analyses.</p

Impairment of alfa signaling in HCVcc-infected hepatocytes.

<p>(A) Naive and HCVcc infected iHLC cultures maintained in presence or absence of the IL28RA nAb were treated for 15 minutes with 10 ng/mL or 100 ng/mL of alfa or Lambda. Cell lysates were then prepared, and equal amounts of proteins subjected to Western immunoblotting to examine the levels of STAT1 phosphorylation using an antibody directed against phospho STAT1 (pSTAT1; Tyr701). Detection of total STAT1 served as loading control to ensure that equivalent amounts of protein were analyzed among samples. (B) Phosphorylation of STAT1 in iHLCs was evaluated upon stimulation using the Luminex bead-based assay. MFI values were reported as mean values of three independent cultures. Error bars show the standard deviations. Two-way ANOVA statistical analysis was performed using Bonferroni post test: ***, <i>P</i> < 0.001.</p

Lambda induces broader and more sustained biological activities than alfa in naive hepatocytes.

<p>Microarray transcriptional profiling of naive iHLCs treated with 10 ng/mL of alfa or Lambda over the course of 48 hours. (A) Relative expression of 83 genes shown to be up-regulated >1.5-fold at all time points by one or both agents, normalized to untreated iHLC at the same time point. Genes indicated by dark blue overlay are consistently regulated by both agents throughout the time series, while genes indicated by light blue overlay show more sustained induction with Lambda compared to alfa. (B) Venn diagram displaying numbers of genes up-regulated (>1.5-fold change) following the indicated treatments. Significance values from MetaCore network analysis for the top six signaling pathways related to the 45 genes with sustained up-regulation by Lambda, and their corresponding <i>P</i> value for the list of 38 shared genes.</p

HCV infection induces transcriptional down-regulation of IFNAR1 in PHH.

<p>Transcriptional profiling of PHH infected with HCVcc at an MOI of 0.2. (A) Relative expression of genes involved in the PAMP recognition pathway showing data from three replicates at 24 h or 48 h post-infection normalized to naive PHH at the same time points. HUGO nomenclature for the genes is to the left. Statistical analysis was performed using one-way ANOVA and <i>P</i> values reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121734#pone.0121734.s006" target="_blank">S1 Table</a> (B) Expression levels of IFNAR1 and IL28RA transcripts in naive (closed circle) and HCVcc-infected (open circle) PHH. The data points represent the RMA values from each individual analyte and horizontal bars indicate the mean levels from three biological replicates. The data shown is representative of two independent experiments.</p

Amino Acid Substitutions in Mutants of the Yeast G-αq Protein Gpa1

<p>A theoretical three-dimensional structure of the yeast Gpa1 G-α protein in complex with the Ste4 protein (G-β) is shown. The position of four amino acid substitutions with phenotypes of interest is indicated by circles. Two alpha helices are indicated by yellow highlighting of the protein backbone. In higher eukaryotes, these helices are considered to form the interface with G-αq downstream effector proteins. Three mutations affecting adaptation to mating pheromone lie on this face: E355K and E364K both hyper-adapt while the M362I allele described in this work is hypo-adaptive. For reference, the position of a mutation affecting sensitivity to RGS GAP activity, G302S, is also shown.</p

Effect of BMS-192364 in Combination with Other Modulators of Calcium Signaling

<div><p>The graphs display fluorescence intensity measurements for HEK293 cells preloaded with Fluo-4 then stimulated with the muscarinic GPCR agonist carbachol at 100 μM. Five baseline fluorescence measurements were taken prior to the injection of carbachol. Where indicated, BMS-195270 or BMS-192364 (100 μM) were added 15 min prior to the carbachol stimulation. The timing of carbachol addition is indicated by a black arrowhead.</p><p>(A) Where indicated, cells were pre-incubated for 15 min with the calcium channel blocker niguldipine (100 μM).</p><p>(B) Where indicated, cells were pre-incubated for 24 h with the G-protein antagonist pertussis toxin (150 ng/ml).</p><p>(C) The treated cells are overexpressing the G-αq mutant allele G188S, which is known to be insensitive to RGS GAP activity.</p></div

Effect of Compounds on Agonist-Induced Calcium Flux

<div><p>(A) The graph displays fluorescence intensity measurements for HEK293 cells preloaded with Fluo-4. Five baseline fluorescence measurements were taken prior to the injection of the muscarinic GPCR agonist carbachol. The timing of agonist addition is indicated by a black arrowhead. Measurements were performed in the presence of vehicle or the BMS small molecules indicated, at 100 μM.</p><p>(B) Dose-response analysis for the effect of compounds on carbachol-stimulated calcium flux. White squares, BMS-195270; EC₅₀ 2 μM. Black squares, BMS-192364; EC₅₀ 9 μM.</p><p>(C) The graph displays fluorescence intensity measurements for primary smooth muscle cells preloaded with Fluo-4. Five baseline fluorescence measurements were taken prior to the injection of the GPCR agonist histamine. The timing of agonist addition is indicated by a black arrowhead. Measurements were performed in the presence of vehicle or BMS-192364 (100 μM).</p></div

Effect of Small Molecules on Wild-Type and Mutant C. elegans

<div><p>(A) The gonad/vulval region of wild-type worms is shown. In the left panel, black arrows indicate the normal, organized array of early stage eggs. The right panel shows a worm treated with BMS-192364 at 0.3 mM. The white arrows indicate late stage eggs that have been retained in the gonad.</p><p>(B) Dose-response curve for BMS-192364, showing effect on egg laying in C. elegans. The percentage of worms displaying an Egl-d phenotype was determined by counting the number of “commas” contained within the animal.</p><p>(C) Quantification of the Egl-d phenotype in four C. elegans mutant strains—<i>ep271, ep272, ep273,</i> and <i>ep275—</i>that were identified in a screen for resistance to the small molecule. Black bars, no treatment. Grey bars, worms treated with BMS-192364 at 0.4 mM.</p><p>(D) Table showing identity of the affected gene in <i>C. elegans–</i>resistant mutant strains, the amino acid changes, and predicted effect on protein function.</p></div

The Yeast <i>gpa1-M362I</i> Mutant Allele Causes a Hypo-Adaptation Phenotype

<div><p>Images show the growth of a monolayer of yeast cells around a paper disc containing alpha factor, the peptide ligand for the Ste3 GPCR. A zone of growth inhibition is visible as a “halo” around each disc.</p><p>(A) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(B) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p><p>(C) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(D) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p></div