HCV cell culture and chemicals
The stable S3-GFP replicon cell line (HCV2a) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, sodium pyruvate, nonessential amino acids, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum supplemented with G-418 (1 μg/mL) . Nile red, sodium oleate, sodium palmitate, and fatty acid free bovine serum albumin (BSA) were obtained from Sigma Chemical Co., Saint Louis, MO. Recombinant human IFN-α 2b (Intron A) was purchased from Schering Plough, Kenilworth, NJ. The Huh-7.5 cell line was obtained from the laboratory of Charlie Rice (The Rockefeller University, New York) and maintained in DMEM with 10% FBS.
Protein lysates from S3-GFP replicon cells were prepared after treatment with FFAs. Equal amounts of protein were resolved on SDS-PAGE gels . The antibodies to Stat1, Stat2, p-Stat1 (Y701), p-Stat2 (Tyr690), p-Jak1 (Tyr1022/1023), p-Tyk2 (Tyr1054/1055), total eIF2α, p-eIF2α (Ser51), beta-actin, IRE1-alpha, PKR, PERK, BIP, SOCS-3, anti-mouse IgG, and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling, Beverly, MA. Antibodies to interferon alpha-receptor 2 (IFNAR2) and p-IFNAR1 were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. The antibody to p-PKR (pT446) was obtained from Epitomics, Burlingame, CA. A mouse monoclonal antibody to IFNAR1 (GB8) was kindly provided by Biogen Idec Inc., Cambridge, MA, USA.
Fatty acid treatment
We used a formulation of FFA mixture at a 2:1 ratio of oleate to palmitate that mimics benign chronic steatosis with low toxicity described by other investigators [22–25]. Briefly, 100 mM palmitate (Sigma catalog No. P-0500) and 100 mM Oleate (Sigma Catalog No. 0–7501) stocks were prepared in 0.1 M NaOH at 70°C and filter sterilized. Five percent (w/v) FFA-free BSA solution was prepared in double distilled water and filter sterilized. A 5 mM stock solution for each fatty acid was prepared in 5% BSA solution in distilled water at 60°C then the mixture was cooled to room temperature. These two FFAs were first mixed together in growth medium in a sterile environment under laminar flow so that the final concentration of FFA was 1 mM and BSA was 1%. S3-GFP cells were cultured in 10-cm dishes to 80-85% confluence and co-cultured with different concentrations of FFA for 24 hours to induce steatosis. At indicated time points, the cells were washed in PBS and cultured in regular growth medium containing 10% FBS.
Nile red staining and microfluorometry
Intracellular fat accumulation in the HCV cell culture was determined by Nile red staining as described . Briefly, hepatocyte monolayers were washed twice with phosphate-buffered saline (PBS) and incubated for 15 minutes with Nile red solution at a concentration of 1 mg/mL in PBS at 37°C. After this treatment, the cell monolayer was washed with PBS and counterstained with a nuclear staining Hoechst dye (H33342, Calbiochem, Darmstadt, Germany) at a concentration of 10 μg/mL prepared in PBS. Cells were then examined under a fluorescence microscope as described previously . The intracellular fat content was quantitated using a microfluorometer (excitation 488 nm and emission 550 nm).
Intracellular fat accumulation in the FFA treated cells was also confirmed by electron microscopy using a standard protocol .
The effect of FFA treatment on the cell proliferation was measured using a MTT colorimetric assay . The assay uses a tetrazolium compound (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) (M5655, Sigma-Aldrich, St Louis, MO), which is reduced intracellularly to formazan by a mitochondrial dehydrogenase enzyme. The percentage of cell viability was determined by comparison with untreated controls.
The effect of FFA treatment on HCV replication and IFNAR1 expression was measured by flow analysis as described previously . Briefly, S3-GFP replicon cells with or without FFA treatment were collected 24 h post- treatment. Intracellular GFP expression was quantified using a flow cytometer (BD LSR II, BD Biosciences). For the analysis of IFNAR1 expression, Huh-7 cells were treated similarly and processed using an IFNAR1 antibody (Epitomics, Burlingame, CA), as per the supplier’s protocol, with the secondary antibody (Alexa Fluor 488-labeled) procured from Invitrogen, CA. Histograms were generated using WinMDI version 2.8 software (Scripps.edu).
Total RNA was isolated from the cells using the GITC method and real-time RT-PCR was carried out as described .
Infectious cell culture model for HCV with a luciferase reporter
Full-length chimeric virus encoding Renilla luciferase (pJFH-ΔV3-Rluc) was kindly provided by Curt H Hagedorn, University of Utah School of Medicine, Salt Lake City . The pJFH-ΔV3-Rluc plasmid was linearized with Xba I, extracted with phenol /chloroform and precipitated with ethanol. HCV-RNA transcripts were prepared in vitro by using T7 RNA polymerase. 1 × 107 Huh-7.5 cells were electroporated with 20 μg of HCV RNA. Transfected cells were cultured in complete DMEM. Infectivity assay was carried out using the supernatants 96 hr post-transfection. Briefly, Huh-7.5 cells were infected with JFH-ΔV3-Rluc virus (MOI 0.1) overnight. On the following day, the infected culture was washed with PBS and then incubated with 10 mL of DMEM with 10% FBS. Infected Huh-7.5 cells were cultured long-term by splitting at a 1:10 ratio at five-day intervals. Replication of HCV in the infected cell culture at each interval was confirmed by measuring the Renilla luciferase activity or the HCV RNA level by RT-qPCR. Renilla luciferase activity was measured in 5μL cell lysate using a luciferase assay kit (Promega) and total protein was measured using the Bradford assay.
The effect of FFA treatment on Jak-Stat signaling and IFN-β (pISRE-Firefly luciferase) promoter activity was examined using a published protocol (21). The ATF6-Luc activity was measured by co-transfecting 0.5 μg of p5XATF6GL3 (Addgene, Cambridge, MA) DNA along with pRL-TK plasmid (0.5 μg) in S3-GFP cells 24 h prior to FFA treatment. Cell lysates were prepared 24 h post FFA treatment and the Firefly luciferase values were normalized to the Renilla luciferase values.
Immunohistochemical staining for HCV-core protein
Infected Huh-7.5 cells were mounted onto a glass slide via the cytospin method. The cells were washed twice with 10 mM PBS pH 7.4 (Sigma-Aldrich, St Louis, MO) for 5 minutes. The cells were fixed in chilled acetone for 15 minutes and then permeabilized by treatment with Reveal Decloaker RTU (Biocare Medical, RV 100) for 25 minutes at boiling point. Slides were then cooled down at room temperature for 25 minutes. Blocking was performed utilizing Background Sniper (Biocare Medical, BS966) for 10 minute at room temperature. The cells were incubated with monoclonal anti-core antibody (Thermo Scientific, Pierce hepatitis C virus core antigen specific mouse monoclonal antibody, Ma1-080) at 1:200 diluted with Da Vinci Green Diluent (Biocare Medical, PD900) for 1 hour at room temperature. Following the primary antibody incubation, the cells were washed 3 times in Tris Buffered Saline (pH 8.0), and incubated with MACH 4 mouse probe (Biocare Medical, UP534) for 10 minutes. After mouse probe treated, the cells were incubated with MACH4 HRP Polymer (Biocare Medical, MRH534) for 30 minutes, and the cells were washed with TBS 3 times. Next, the cells were treated with diaminobenzidine (DAB) chromogen (Dako Cytomation, Carpinteria, CA) for 5 minutes. The slides were counterstained with hematoxylin for 30 seconds and Tacha’s bluing Solution (Biocare Medical, HTBLU) for 30 seconds, dehydrated, mounted and observed by light microscopy.
Microfluorometry assay results, ISRE, ATF6 (activating transcription factor 6), and HCV (pJFH-ΔV3-Rluc) luciferase activity, real time RT-PCR and FACS analysis were compared for significance using the Student’s t test. P values less than 0.05 were considered significant.