Jak inhibitor and siRNA targeted to Stat2 prevent IFN-λ antiviral mechanisms against HCV in FFA treated cell culture.

<p>(<b>A and B</b>). FFA treated HCV culture is treated with Jak1 inhibitor for seven days. Antiviral activity of IFN-αand IFN-λwas determined each day by Renilla Luciferase. (<b>C</b>). FFA treated HCV cell culture was treated with siRNA targeting Stat1, Stat2 and Stat3 and after 24 hours culture was treated with IFN-λ. After 72 hours antiviral effect of was determined by Luciferase assay. Silencing Stat2 only prevent IFN-λantiviral activity against HCV in FFA treated culture.</p

IFNAR1 binds to LAMP2A and localizes to CMA-associated lysosomes.

<p>(<b>A</b>). Co-immunoprecipitation assay of Huh-7.5 cells serum starved for six hours, after which cells were lysed, and lysates were immunoprecipitated with either IFN-λ receptor or IFN-α receptor and probed for LAMP2A or HSC70 by Western blot analysis. (<b>B</b>). Effect of CMA on IFNAR1 localization with LAMP2A. Confocal images of cellular localization of IFNAR1 (green) and LAMP2A (red) in Huh-7.5 cells cultured with serum free medium. DAPI was used for nuclear stain. Only serum starvation enhances CMA, which is associated with increase signal co-localization (red+green = yellow) indicating that IFNAR1 localizing to CMA associated lysosomes for degradation. (<b>C</b>) Effect of CMA on IFN-λ receptor localization with LAMP2A. There was no increase in signal co-localization.</p

Persistently infected Huh-7.5 cells were treated with 1xIC₉₀, 2.5 × IC₉₀, and 5 × IC₉₀ IFN-α or IFN-λ.

<p>After 72 hours antiviral efficacy of IFN-α and IFN-λ was determined by the measurement of <i>Renilla</i> luciferase activity and immunohistochemistry for HCV Core protein expression. <b>(A).</b> Normalized <i>Renilla</i> luciferase activity of culture treated with equivalent concentrations of IFN-alpha or IFN-λ. <b>(B).</b> Representative picture showing the expression of HCV core protein in the IFN-treated cells and control uninfected Huh-7.5 by immunostaining. <b>(C).</b> Quantification of HCV Core⁺ cells in 10 different high-power fields (×40), compared with untreated control. Both assays confirmed that the antiviral effect of IFN-λ is significantly stronger than IFN-α when used at equivalent concentrations (* p< 0.03, *** p< 0.001).</p

FFA treatment for 48 hours induced decrease of IFNAR1 in Huh-7.5 cells is mediated by a lysosomal degradation pathway.

<p>Huh-7.5 cells were treated with FFA for 24 hours in the presence or absence of lysosome inhibitors such as: NH4Cl (30mM) or BafilomycinA1. The expression levels IFNAR1, IL10Rβ and β-actin were examined using the cell lysates by Western blotting.</p

FFA treatment for 48 hours induced autophagy response in Huh-7.5 cells.

<p>Huh-7.5 cells were treated with increasing concentrations of FFA and after 72 hours and examined for autophagy induction by immunostaining and Western blot analysis. (<b>A)</b> Immunostaining showing expression of p62 in FFA treated Huh-7.5 cells. (B). Western blot show that p62 levels are decreased in FFA treated cells. ATG5 level are induced in FFA treated Huh-7.5 cells. LAMP2A and HSC70 levels and b-actin levels were not altered significantly as compared to untreated Huh-7.5 cells. (C). The expression of p62 was decreased by both HCV and FFA treated culture. The expression of ATG5 and LAMP2A was induced by HCV infected and FFA treated Huh-7.5 cells.</p

Chaperone induced autophagy targets the degradation of IFNAR1 in HCV and FFA treated Huh-7.5 cells for 48 hours.

<p><b>(A).</b> Schematic representation of IFNAR1 protein where presence of CMA motif site is marked. <b>(B).</b> Uninfected Huh-7.5 cells were cultured in a serum free medium for indicated time points. The expression of IFNAR1 and IFNLR1 as well as CMA proteins (LAMP2A and HSC70) levels were measured by Western blotting. <b>(C).</b> Show the dose-dependent reduced expression of IFNAR1 in the Huh-7.5 cells treated with 6-aminonicotinamide (mM) by Western blotting. The expression of IFN-λ receptor did not change by similar treatment. (<b>D</b>). Measurement of IFNAR1 expression after silencing LAMP2A. Huh-7.5 cells were transfected with 60pmole of siRNA using lipofectamine. After 24 hours, cells were serum starved for 4 to 48 hours and cell lysates were measured for IFNAR1 expression by Western blot analysis.</p

FFA treatment for 48 hours results in down-regulation of cytosolic and membrane expression IFNAR1 in Huh-7.5 cells.

<p><b>(A).</b> Western blot showing downregulation of cytosolic low molecular weight IFNAR1 as well as mature high molecular weight IFNAR1 in Huh-7.5 cells treated with FFA in a concentration dependent manner. The expression of IFN-λ1 and IFN-λ receptor was not altered. <b>(B).</b> Expression of cytosolic as well as membrane IFNAR1, IFNGR and IFNLRs in FFA treated Huh-7.5 cells. <b>(C).</b> The surface expression of IFNAR1, IFNGR and IFNLR1 in FFA treated Huh-7.5 cells by confocal microscopy.</p

HCV replication induces microvesicular steatosis in persistently infected Huh-7.5.

<p>(<b>A).</b> Confocal microscopy showing increased accumulation of lipid droplets in Huh-7.5 cells with or without HCV infection. Red: Oil-red staining and green: HCV core expression. The images were taken at 40X magnification. (<b>B).</b> Microfluorometer analysis of intracellular fat accumulation in Huh-7 cells with or without HCV replication. The OD values are compared between uninfected and HCV infected cells. (<b>C).</b> Electron micrograph showing more intracellular lipid droplet accumulation in persistently HCV infected as compared to uninfected Huh-7.5 cell. (<b>D).</b> FFA treatment induces macrovesicular steatosis in Huh-7.5 cells. Uninfected Huh-7.5 cells were treated with increasing concentration of Oleate/Palmitate (2:1 ratio) for 24 hours and hepatocellular steatosis was examined by confocal microscopy after Oil-red staining. (<b>E</b>). Microfluorometer analysis shows dose-dependent intracellular fat accumulation in Huh-7.5 cells treated with oleate and palmitate (FFA). OD values are compared between untreated and treated cells. <b>F.</b> FFA treatment support HCV replication in cell culture. Huh-7.5 cells were infected with JFH1-ΔV3-Rluc and then treated with FFA 0.5mM. Replication of HCV was measured by Renilla luciferase activity.</p