Volume 143, Issue 2, Pages 429-438.e8 (August 2012) Persistence of HCV in Quiescent Hepatic Cells Under Conditions of an Interferon- Induced Antiviral Response  Oliver Bauhofer, Alessia Ruggieri, Bianca Schmid, Peter Schirmacher, Ralf Bartenschlager  Gastroenterology  Volume 143, Issue 2, Pages 429-438.e8 (August 2012) DOI: 10.1053/j.gastro.2012.04.018 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Differentiation and polarization of Huh7.5 cells. (A) Morphologies of naïve or DMSO-treated Huh7.5 cells. Days of culture are specified in the top. After 14 days of DMSO treatment, cells were growing in monolayers (a) and ridge-like structures (b). Within the ridges, distinct areas were speared appearing as “holes” in the cell layer (c; arrow). (B) Immunofluorescence of Huh7.5dif cells after 14 days of culture. Plasma membrane was visualized by staining F-actin with Alexa Fluor 646–conjugated phalloidin; canaliculi were stained with an MRP2-specific antibody. (C) Expression of markers of hepatocyte differentiation in Huh7.5 cells 3, 7, and 14 days after cultivation in DMSO-containing medium as determined by qRT-PCR. mRNA amounts in cells cultivated in DMSO-free medium (control) were used for normalization (*P < .02). PHHs and human liver samples served as reference. (D) Impact of HCV infection and IFN-α treatment on expression of differentiation markers. Huh7.5 cells were differentiated by 14-day cultivation in DMSO-containing medium and cultured for 28 further days under conditions specified. mRNA amounts in cells cultivated in DMSO-free medium (control) were used for normalization. Means from 3 measurements and SDs are shown (*P < .01). Differences between individual samples were all nonsignificant. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Replication and spread of HCV in Huh7.5dif cells. (A) Cells were infected with HCV. At the time points specified, infectivity titers in supernatant and intracellular HCV RNA amounts were determined by limiting dilution assay and qRT-PCR, respectively. Mean values from 5 independent experiments and SDs are shown. (B) Huh7.5dif cells were infected with Jc1 and fixed at indicated time points, and NS5A was detected by immunofluorescence microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) to determine total cell numbers. For quantification (bottom panel), 5000–8000 cells in 5 view fields of 4 independent experiments were counted. Mean values of 4 independent experiments and their respective SDs are shown. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Persistence of HCV in IFN-α–treated Huh7.5dif cells. (A) Schematic of the experimental setup. (B) Infectivity titers in the supernatant of Huh7.5dif cells cultured in the absence or presence of IFN-α as specified in A. Standard box blots (median, 25th and 75th quantiles) are shown. The whiskers represent the 5th and 95th percentiles; they are not visible due to low variability. Data are the mean from 5 independent experiments and their respective SDs. (C) Intracellular HCV RNA amounts in infected Huh7.5dif cells that were cultured in the presence or absence of IFN-α. Cells were harvested at the time points specified and viral RNA was quantified by using qRT-PCR. (D) Infected Huh7.5dif cells cultured in the presence or absence of IFN-α were fixed at given time points and processed for NS5A-specific immunofluorescence. Nuclei were stained with DAPI to determine total cell numbers. For quantification (bottom panel), 5000–8000 cells in 5 view fields of 4 independent experiments were counted. Mean values from 4 independent experiments are shown by standard box blots. The whiskers represent the 5th and 95th percentiles. Outliers are depicted by dots. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Stochastic nature of the IFN response. (A, left panel) Huh7.5dif cells were infected with HCV or mock infected and treated with 100 IU/mL IFN-α. Cells were fixed at indicated time points and stained for MxA. Representative panels are shown for 2 and 28 days of IFN-α treatment. MxA-negative cells are indicated by arrows (original magnification 20×). Note the heterogeneity of MxA expression, which is independent from HCV infection. A quantification of the experiment is shown in the right panel. Cells were analyzed by microscopy for a detectable MxA signal, and cell numbers were determined by counting DAPI-stained nuclei. For each time point, 5 view fields, each from 2 independent experiments, were counted. Mean values of the 10 view fields are shown. Absolute numbers of HCV-positive cells are given in the respective bars. (B) Huh7.5-MxAdeGFP cells were stimulated with the IFN-α concentrations specified, sorted into GFP-negative and -positive populations, replated, and restimulated with the IFN-α concentrations given on the right. The percentage of GFP-positive cells is given. (C, left panel) PHHs infected for 24 hours with 5 TCID50 of Jc1/cell or control cells were treated with 100 IU/mL IFN-α for 16 hours or left untreated, fixed, and stained for MxA. Representative view fields are shown. MxA-negative cells are indicated by arrows. Attempts to detect HCV antigen were not successful due to high background. (C, right panel) MxA amounts were quantified in 3 view fields. Background staining not associated with cells was excluded. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 HCV replicates preferentially in IFN-low responding cells. (A) Huh7.5dif cells were infected with HCV and cultured in medium containing 100 IU/mL IFN-α. Cells were fixed 28 days later and NS5A and MxA were detected by immunofluorescence (left panel). Boxed and labeled areas in the merge picture are shown as enlargements at the top. Quantification of 2 independent experiments conducted as explained for Figure 4A is shown in the middle panel. Absolute numbers of HCV-positive cells in both experiments are given in the respective bars. In the right panel, MxA and NS5A double-positive cells were assessed for their NS5A levels and categorized into low- or high-level NS5A-expressing cells. (B) Huh7.5-MxA or Huh7-IFIT1deGFP cells were stimulated with IFN-α and 24 hours later sorted into GFP-positive and GFP-negative populations by flow cytometry. Sorted cells were replated and infected with HCV, and 72 hours later cells were fixed and stained for NS5A by immunofluorescence. The percentage of NS5A-positive cells is given. (C) PHHs transduced with a sensitive fluorescent reporter to detect HCV replication (red fluorescent protein [RFP] accumulating in the nucleus of HCV-infected cells36) were infected with HCV and treated with 100 IU/mL IFN-α (left panel). Cells were fixed and stained for MxA by immunofluorescence. Representative images of PHHs obtained from 2 different donors are shown. Noninfected or infected cells (corresponding to cytosolic or nuclear RFP, respectively) are marked with black or white arrows, respectively. Quantification of MxA-specific signal in HCV-infected cells (right panel). For each condition, at least 10 cells were analyzed. Shown are the mean values with the 95% confidence interval (*P < .01). Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 HCV replication relapses upon IFN-α removal. (A) HCV-infected Huh7.5dif cells were treated with 100 IU/mL IFN-α for 28 or 35 days or left untreated (control). In the experiment represented by the right bar, cells were treated for 28 days with IFN-α and then cultured for 7 days in IFN-free medium. Culture supernatants were harvested and titers of infectious HCV contained therein were determined by limiting dilution assays. Shown are the means from 5 independent experiments and SDs. Cells described in A were harvested, total RNA was extracted, and amounts of (B) HCV RNA and (C) MxA RNA were determined by qRT-PCR. Values were normalized to infected cells without IFN treatment. Mean values from 5 independent experiments are depicted by box plots as described in Figure 3C. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Comparison of antiviral potency of IFN-α and IFN-λ in Huh7.5dif cells. Huh7.5dif cells were infected with Jc1 and 48 hours later treated with IFN-α or IFN-λ at their respective IC90s (10 IU/mL IFN-α; 1 ng/mL IFN-λ) as determined with nondifferentiated cells. At indicated time points, supernatants were harvested and used to determine (A) HCV infectivity titers, (B) intracellular HCV RNA amounts, and (C) MxA mRNA amounts. Values in A–C correspond to mean values of 4 independent experiments. For B and C, the mean value with the respective SD is shown. (D) Cell surface abundance of IFN receptors (IFNAR1, IFNAR2, and IL28Rα) was detected by flow cytometry in naïve Huh7.5 and Huh7.5dif cells. (E) Cells described in D were lysed, total RNA was extracted, and mRNA amounts of the genes specified were determined by qRT-PCR. Shown is the fold change as determined with naïve (nondifferentiated) cells that was set to 1. Mean values and SDs from 3 independent experiments are depicted in D and E. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Comparison of replication kinetics in naïve and differentiated Huh7.5 cells. (A) Huh7.5 cells were infected with 1 TCID50 of Jc1/cell and 6 hours later washed extensively with medium. One, 3, 6, and 8 days later, supernatants were harvested and infectivity titers were determined by limiting dilution assay. In addition, cells were lysed and HCV RNA content was measured by qRT-PCR. (B and C) Huh7.5dif cells were infected with Jc1 at 1 TCID50/cell. Cells were then either treated with 100 IU IFN-α/mL or left untreated, and 0, 2, 4, and 8 days posttreatment cells were lysed and supernatants were harvested. (B) HCV RNA in cell lysates and (C) infectivity titers in the supernatant were determined by qRT-PCR or limiting dilution assay, respectively. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Characterization of the BAC-transduced Huh7.5-MxAdeGFP cell line. (A) Schematic of the BAC constructs. Destabilization of the MxA- or IFIT1-deGFP fusion protein was achieved by C-terminal fusion with the degradation domain of mouse ornithine decarboxylase, reducing the half-life of the fusion protein to ∼3.5 hours. (B) Western blot of cell extracts from Huh7.5-MxAdeGFP cells showing the simultaneous expression of endogenous MxA and MxAdeGFP on 17- to 39-hour stimulation with 100 IU/mL IFN-α. (C) Huh7.5-MxAdeGFP cells were treated with 100 or 1000 IU/mL IFN-α for indicated periods, fixed, and analyzed for expression of MxAdeGFP or endogenous MxA by using flow cytometry. The percentage of MxAdeGFP- or MxA-positive cells is shown. (D) Huh7.5-MxAdeGFP cells were treated with 1000 IU/mL IFN-α and sorted as described (left panel). The fluorescence-activated cell sorter dot blot displays the MxAdeGFP-positive and -negative cell populations used for the analysis shown in the right panel. Directly after sorting, cells were lysed and total RNA was extracted. Amounts of the mRNAs specified were quantified by using qRT-PCR (right panel). Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Determination of the IC90 for IFN-α and IFN-λ in continuously growing nondifferentiated Huh7.5 cells and comparison of the antiviral potency of both cytokines. (A) Cells were infected with the JcR2a reporter virus for 24 hours with 2 TCID50/cell. This virus contains a Renilla luciferase gene that is inserted into the HCV genome and thus can be used to directly quantify viral RNA replication.5 IFN-α or IFN-λ were added at increasing concentrations to the culture medium, and 72 hours later cells were lysed and luciferase activity was measured. Shown are the mean values and SDs of a representative experiment out of 6 independent repetitions. IC90 values determined in this assay are 10 IU/mL for IFN-α and 1 ng/mL for IFN-λ, respectively. (B) Huh7.5 cells infected with Jc1 at 1 TCID50/cell were treated with IFN-α or IFN-λ at their respective IC90s (10 IU/mL and 1 ng/mL, respectively), and 0, 2, 5, and 7 days after the start of the treatment supernatants were harvested and infectivity titers were determined. (C) Differentiated Huh7.5 cells were infected with Jc1 at 1 TCID50/cell for 2 days. Cells were then treated with 100 IU/mL IFN-α or 10 ng/mL IFN-λ, respectively (each corresponding to 10× IC90). Zero, 2, 5, and 7 days after treatment, release of infectious particles was measured by limiting dilution assay. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Impact of differentiation on IFN-α– and IFN-λ–mediated ISG induction. Cells specified at the top were treated with 10 IU/mL IFN-α or 1 ng/mL IFN-λ, each corresponding to the IC90 of HCV replication as determined with a standard culture system (Supplementary Figure 3) or left untreated (naïve). Cells not treated with IFN were cultured in parallel. After 16 hours, cells were lysed and total RNA was extracted and used for transcriptome analysis. Data were evaluated by using the Chipster software package. Depicted are all ISGs with at least 2-fold changes of expression level as compared with mock-treated cells. White boxes indicate genes for which changes of expression levels were less than 2-fold. A summary of all the gene names and measured values is given in Supplementary Table 1. Gastroenterology 2012 143, 429-438.e8DOI: (10.1053/j.gastro.2012.04.018) Copyright © 2012 AGA Institute Terms and Conditions