TLR3 expressed in 293-hTLR3 cells senses HCV replication in adjacent human hepatocytes.

<p>(<b>A</b>) Experimental design, showing co-culture of HCV-nonpermissive, TLR3-competent 293FT/IFN-β-mCherry cells expressing mCherry under control of the IFN-β promoter with Huh-7.5 cells that are HCV permissive and TLR3 incompetent. Cells were infected with HJ3-5/5A-YFP virus that expresses YFP as a fusion with NS5A. (<b>B</b>) Immunofluorescence microscopy demonstrating induction of mCherry expression in 293FT/IFN-β-mCherry + Huh-7.5 cell co-cultures upon stimulation with poly(I:C) or infection with HJ3-5/NS5A-YFP virus. HCV replication was visualized by YFP expression and present in cells adjacent to those expressing mCherry in the two-color merged images at the far right. Nuclei were visualized by DAPI counterstain. (<b>C</b>) siRNA-mediated depletion of TLR3 significantly reduces poly-(I:C)-induced activation of the IFN-β promoter in 293FT/IFN-β-mCherry cells. (<i>left</i>) Fold-change in luciferase activity induced by extracellular poly-(I:C) in 293FT/IFN-β-mCherry cells transfected with TLR3-specific (siTLR3) or control (siCont) siRNAs. The cells were transduced with an IFN-β-Luc reporter plasmid. * p≤0.02. (<i>right</i>) qRT-PCR detection of TLR3-specific mRNA in cells transfected with the siTLR3 or siCont siRNAs. * p≤0.02. (<b>D</b>) Poly-(I:C)-induced expression of mCherry in 293FT/IFN-β-mCherry cells is ablated by prior transfection with TLR3-specific siRNA. (<b>E</b>) siRNA-mediated depletion of TLR3 eliminates mCherry expression by 293FT/IFN-β-mCherry cells placed in co-culture with HCV-infected Huh-7.5 cells. 293FT/IFN-β-mCherry cells were transfected with siTLR3 or siCont siRNAs 3 days prior to being placed in co-culture with Huh-7.5 cells infected with genotype 2a JFH-1 virus. (<b>F</b>) Experimental design, showing Huh7.5-TLR3 cells co-cultured with Huh7.5 cells supporting replication of a genome-length genotype 1a HCV RNA replicon. (<b>G</b>) Co-immunoprecipitation analysis of the association of HCV RNA with Flag-TLR3 in co-cultures of Huh7.5-TLR3 and HCV replicon cells (panel F) after treatment with bafilomycin (1.0 nM, lanes 2 and 5) or chloroquine (5.0 µM, lanes 3 and 6) for 72 hrs. RNA was extracted from anti-Flag precipitates prepared from lysates of the co-cultured cells and subjected to HCV-specific RT-PCR. See legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003345#ppat-1003345-g001" target="_blank">Fig. 1E</a> for further details.