Intracellular expression of STAT1-CC induces p-PKR and p-eIF2α in the resistant cell line affer IFN-γ treatment.

<p>The IFN-γ resistant cell line (GR17-1) was transfected with each STAT1 expression plasmid and treated with or without IFN-γ. At 24 hours post-transfection protein lysates were obtained and examined for p-PKR and p-EIF2α levels by western blot analysis. A protein lysate from the S9-13 cell line was obtained 30 minutes after IFN-γ addition and used as a positive control.</p

STAT1-CC plus IFN-γ transfected resistant (GR17-1) cells display a marked reduction in HCV NS3 protein.

<p>IFN-γ resistant cells were transfected separately with the respective STAT1 plasmid and treated with or without IFN-γ. At 72 hours the cells were mounted onto a glass slide, stained for with DAB for HCV NS3 (brown), and counterstained with hematoxylin (blue). Huh7 cells without virus were used as a negative control.</p

IFN-γ dependent activation of GAS promoter by the STAT1-CC molecule in the resistant (GR17-1) replicon.

<p>(<b>A</b>) Summary of different modified STAT plasmid constructs used in this project. (<b>i</b>) Shows pRC-CMV plasmid containing human full-length wild type STAT1 cDNA, STAT1-CC containing two cysteine substitutions in the SH2 Domain of STAT1 at residues 656 and 658 and STAT1-CC-Y701F also contained the double cysteine substituted residues plus a phenylalanine substitution at residue 701. (<b>ii</b>) Shows the pRC-CMV expression plasmid containing the full-length human wild type STAT3 cDNA. STAT3-CC double cysteine substituted residues in the SH2 Domain of STAT3 at residues 661 and 663, STAT3-CC-Y705F also contained the double cysteine substituted residues plus a phenylalanine substitution at residue 705. (<b>iii</b>) STAT1 constructs with a C-terminal GFP fusion, STAT1-CC-GFP and STAT1-CC-Y701F–GFP plasmid. (<b>iv</b>) Firefly luciferase reporter construct driven by GAS promoters. (<b>B</b>) Show the activation of GAS promoter in resistant cell line transfected with Stat1, Stat1CC and Stat1CC Y-F plasmids. IFN-γ resistant cells (GR17-1) were transiently co-transfected with a GAS-firefly luciferase reporter, the indicated STAT1 construct and a control plasmid expressing renilla luciferase. Firefly luciferase activity was normalized with the transfection control renilla luciferase. Each bar represents the fold increase in GAS promoter expression activity at 24 hours after the addition of IFN. The error bars represent the SEM from six independent experiments. GAS-firefly luciferase activity increased in response to IFN-γ treatment. IFN-α treatment did not induce the GAS promoter. (<b>C</b>) Shows that the activation of the GAS promoter is specific to STAT1-CC. Interferon resistant cells (GR17-1) were transiently co-transfected with a GAS luciferase reporter and one microgram of each of the constructs using the FuGENE 6 reagent. Values were normalized with renilla luciferase and presented as the fold increase in GAS promoter induction between IFN naïve and IFN-γ treated cells 24 hours post-transfection are shown. (<b>D</b>) Dose dependent activation of the GAS promoter by STAT1-CC molecules in the GR17-1 cell line. The STAT1 constructs induce the GAS promoter in a dose dependent manner. Different concentrations (0.5, 1, and 2 µg) of the respective STAT1 plasmid and 0.5 µg of GAS-luciferase were co-transfected to GR17-1 cells by FuGENE 6 reagent and then treated with 1000 IU of IFN-γ. At 24 hours, the luciferase activity was measured and normalized with renilla luciferase as a transfection control. Values represent normalized luciferase expression (RLU) from three experiments. (<b>E</b>) Prolonged GAS-luciferase activity in the resistant cells transfected with STAT1-CC compared to wild type STAT1. At the indicated time points, RLU activity was measured, normalized with renilla luciferase, and represented as fold change in GAS induction after IFN-γ addition.</p

RPA assay demonstrates that STAT1-CC plus IFN-γ eliminates HCV negative strand RNA in resistant replicon cells.

<p>IFN-γ resistant (GR17-1) cells were transfected with the respective plasmid and treated with or without IFN-γ. At 72 hours post-transfection total RNA was isolated by the GITC method and subjected to RPA analysis for HCV negative strand RNA using a probe targeted to the highly conserved 5′-UTR region. The GAPDH mRNA was used as loading control. The nucleotide number in each blot shows the length of the protected fragment.</p

Intracellular STAT1-CC upregulates HLA class I surface expression in IFN-γ resistant (GR17-1) cells.

<p>GR17-1 cells were transfected with STAT1, STAT1-CC or STAT1-CC-Y701F plasmid using FuGENE 6 reagent for 72 hours with or without IFN-γ treatment. After 72 hours, the cells were harvested and stained with a phycoerythrin-conjugated anti-human HLA antibody. Surface HLA-1 expression was then quantified by flow cytometry. (<b>A</b>) Each histogram is representative of six independent experiments. The red cell population in each histogram represents IFN-γ naïve cells and the blue cell population represents IFN-γ treated cells. (<b>B</b>) Mean fluorescence intensity of six independent experiments per group. Values represent fold increase in HLA1 expression after the addition of IFN-γ. Mock represents GR17-1 cells without plasmid transfection. Asterisk (*) denotes a significant (p<0.05) increase in HLA1 in STAT1-CC transfected cells plus IFN-γ compared to mock.</p

Confirmation of the IFN-γ resistance of HCV 1b replicon cell lines.

<p>(<b>A</b>) The GAS-luciferase activity in the sensitive and resistant replicons. One sensitive and eight resistant cell lines were seeded in 24 well plates. The next day the cells were transfected with 1 µg of GAS-firefly luciferase and 500 ng of renilla luciferase plasmid using the FuGENE 6 transfection reagent and then treated with or without IFN-γ (1000 IU/ml). The cells were then assayed for GAS promoter induction 24 hours after the addition of IFN-γ. The GAS firefly luciferase value of each well was normalized with a renilla luciferase control. The values were expressed as fold change in GAS luciferase expression after IFN-γ addition. Error bars represent Standard Error of the Mean (SEM) from six independent experiments. (<b>B</b>) RPA for negative strand RNA showing the replication of HCV in the resistant replicon with and without IFN-γ after 72 hours. Two IFN-γ resistant cell lines were treated with or without IFN-γ (1000 IU/ml). At 72 hours post-transfection total RNA was isolated by the GITC method and subjected to RPA for detection of the positive sense strand of the HCV genotype 1b 5′UTR. (<b>C</b>) Immunostaining of NS3 proteins of HCV using GR17-1 cells treated or not treated with IFN-γ for 72 hours. The IFN-γ resistant GR17-1 cells were treated with or without IFN-γ. At 72 hours the cells were mounted onto a glass slide, stained for HCV NS3 (DAB), and counterstained with hematoxylin.</p

Comparison of spontaneous and IFN-γ dependent phosphorylation of STAT1-CC molecule in sensitive (S9-13) and resistant (GR17-1) cells.

<p>Cells were transfected with individual plasmid constructs of either STAT1-GFP, STAT1-CC-GFP, or STAT1-CC-Y701F–GFP. IFN-γ was added to the appropriate groups at 72 hours post-transfection. Thirty minutes after IFN-γ addition, the protein lysates were immunoprecipitated using an anti-GFP antibody. Immunocomplexes were detected using an antibody to phospho-STAT1 by western blot analysis. <b>Upper panel:</b> Preferential phosphorylation of STAT1-CC over the STAT1 molecule in IFN-γ sensitive Huh-7 cells (S9-13). IFN-γ dependent phosphorylation was seen for both wild type STAT1 molecule as well as Stat1CC molecule. <b>Lower panel:</b> The STAT1-CC showed low level phosphorylation of Stat1CC in the resistant cells in IFN-γ treated cells.</p

Effect of STAT1-CC and IFN-γ on cell culture grown full-length HCV.

<p>Stable sensitive (S5-15) and resistant (GR17-1) cell lines with or without STAT1 or STAT1-CC were infected with full length HCV genotype 2a clone JFH1 virus at MOI of 1.0. After 72 hours cells were treated with IFN-γ. At 72 hours post-infection total RNA was isolated, positive strand HCV RNA level was measured by real-time RT-PCR. (<b>A</b>) Antiviral effect of IFN-γ and IFN-α against cell culture grown full-length HCV. (<b>B</b>) Demonstrate the antiviral effect of Stat1-CC expression on cell culture derived full-length HCV RNA in sensitive and resistant cells due to IFN-γ treatment. Bars are representative of three independent experiments and error bars represent standard error of the mean. Significance was considered at values below 0.05 (p<0.05, student t-test). Asterisks (*) represent significant (p<0.02) reductions in HCV RNA.</p

STAT1-CC-GFP translocates to the nucleus of GR17-1 cells in an IFN-γ dependent manner.

<p>IFN-sensitive (S9-13) and IFN-resistant (GR17-1) cells were transfected with plasmid containing STAT1-GFP, STAT1-CC-GFP and STAT1-CC-Y701F-GFP. The nuclear translocation of each STAT1-GFP construct with or without IFN-γ treatment in the sensitive and resistant cells was examined under a confocal microscope. (<b>A</b>) Shows high resolution picture of Stat1-GFP nuclear translocation after IFN-γ treatment in the sensitive Huh-7 (S9-13) cells. The images are represented as the superimposition of Green Fluorescent Protein (green), To-Pro3 633 (far red), and the differential interference contrast images (DIC) (gray scale). (<b>B</b>) Shows the differential nuclear translocation of STAT1-GFP, STAT1-CC-GFP and STAT1-CC-Y701F-GFP protein in the sensitive (S9-13) versus resistant Huh-7 (GR17-1) cells before and after treatment with IFN-γ. Fluorescence green and red microscopic picture of the same area were taken and superimposed using Abode Photoshop.</p

The cytotoxicity of intracellular expression of each STAT1 molecule in the resistant cells.

<p>The GR17-1 cell line was transfected with each of the STAT1 constructs and the cellular toxicity was performed at 48 hours post-transfection by the MTT assay. The values represent viable cells as a percent of cells plus FuGENE-6 control. Bars represent SEM from three experiments.</p