TLR3 senses double-stranded RNA products of HCV replication in human hepatocytes.

<p>(<b>A</b>) Quantitative RT-PCR analysis of (left) IFN-β and (center and right) ISG56 mRNA in Huh7.5-TLR3 cells infected with HCV. Cells were infected with HCV (HJ3-5) or UV-inactivated HCV at an m.o.i. of 1 for 72 hrs, prior to extraction of RNA and measurement of IFN-β and ISG56 mRNA. Statistical comparisons were made between mock- and HCV-infected cultures (left and center panels), or between samples indicated by bars (right panel): * p≤0.02, ** p≤0.001. (<b>B</b>) Agarose gel electrophoresis of in vitro transcribed HCV RNAs (top panel) before or (bottom panel) after S1 nuclease treatment. M: DNA standards ladder; lane 1, positive-strand HCV RNA; lane 2, negative-strand HCV RNA; lane 3, annealed double-stranded HCV RNA. (<b>C</b>) Quantitative RT-PCR analysis of ISG56 mRNA in (left) PH5CH8 and (right) Huh7.5/TLR3 cells treated with 50 µg/ml of ssRNA or dsRNA for 6 hrs prior to extraction of RNA. Statistical comparisons were made with cultures not treated with RNA: * p≤0.02, ** p≤0.001. (<b>D</b>) (left panel) Immunoblot analysis of ISG15 expression induced by synthetic HCV dsRNA in PH5CH8 cells previously transduced with lentivirus vectors expressing non-targeting (NT) or TLR3-specific shRNAs. β-actin was included as a loading control. (right panel) Similar immunoblots of Huh7.5-TLR3 cells treated with HCV dsRNA, either with or without prior S1 nuclease digestion. In both panels, cell lysates were prepared for immunoblotting 24 hrs after addition of dsRNA. (<b>E</b>) HCV RNA co-immunoprecipitates with Flag-TLR3 from lysates of HCV-infected Huh7.5-TLR3 cells. (top panel) Agarose gel electrophoresis showing detection of HCV-specific RNA by RT-PCR in anti-Flag (lanes 2, 4 and 6), or isotype control (mouse IgG₁, lanes 1, 3 and 5), immunoprecipitates prepared from lysates of Huh7.5-TLR3 cells (lanes 1 and 2), Huh7.5-H539E cells (lanes 3 and 4), or Huh7.5-H539E cells (lanes 5 and 6), infected with HJ3-5 virus (m.o.i. = 1) for 72 hrs. RNA extracted from mock or HCV-infected Huh-7.5 cells was assayed in parallel in the RT-PCR reaction as negative and positive controls, respectively (lanes 7 and 8). (bottom panel) Anti-Flag immunoblots of the respective immunoprecipitates.