14 research outputs found

    Poly(I:C) induced TLR3 signaling is dependent upon a conserved collagen superfamily domain in MSR1.

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    <p>(<b>A</b>) (top) Domain architecture of the 451 a.a. human MSR1 protein showing the location of the collagen superfamily domain that contains multiple G-X-Y repeats that form a triple helix <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003345#ppat.1003345-MarchlerBauer1" target="_blank">[58]</a>. (bottom) Alignment of human MSR1 with other mammalian MSR1 proteins near the C-terminus of the collagen domain, showing multiple conserved basic residues. GenBank accession numbers are shown for each. “Macscav” = conserved macrophage scavenger receptor domain; “SRCR” = Cys-rich scavenger receptor superfamily domain. (<b>B</b>) Myc-MSR1 mutants constructed within the pCX4bsr/Myc-MSR1 plasmid. (<b>C</b>) Poly(I:C) stimulated IFN-β promoter activity in MSR1-depleted Huh7.5 TLR3/shMSR1 cells transiently expressing wt or mutant Myc-MSR1: (left) Myc-MSR1/Δ321–339 (deletion of underlined sequence in panel A), (center) Myc-MSR1/3KA, or (right) Myc-MSR1/R325A and Muc-MSR1/R3KA. Cells were co-transfected with Myc-MSR1 expression vectors, pIFN-β-Luc and pRL-CMV (internal control), cultured for 24 h, then treated with poly(I:C) (50 µg/ml) for 6 hrs before to lysis and luciferase assay. (<b>D</b>) Immunoblots showing expression of wt Myc-MSR1 and related mutants in PH5CH8/shMSR1 cells. Lysates were precipitated with anti-Myc antibody prior to anti-Myc immunoblot. (<b>E</b>) Flow cytometry histograms showing expression of Myc-MSR1 (wt) and indicated mutants on the surface of PH5CH8 cells (green lines). Purple curves indicate cell-surface staining with isotype immunoglobulins. “FL2-H” = fluorescent intensity. (<b>F</b>) Co-immunoprecipitation analysis of the association of HCV RNA with wt versus mutant Myc-MSR1 proteins in PH5CH8/shMSR1 cells co-cultured with Huh7.5 cells infected with HJ3-5 virus (m.o.i. = 1) for 72 hrs. RNA was extracted anti-Myc precipitates from lysates of co-cultures of infected Huh7.5 cells and PH5CH8/shMSR1 cells stably transduced with empty vector (lane 1 and 5), wt Myc-MSR1 (lane 2 and 6), Δ321–339 (lane 3 and 7), or R3KA (lane 4 and 8). 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.</p

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

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    <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 IgG<sub>1</sub>, 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.</p

    MSR1-dependent TLR3 sensing of HCV infection in neighboring cells restricts viral replication.

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    <p>(<b>A</b>) (<i>left</i>) Design of co-culture experiments in which HCV-nonpermissive, but TLR3-competent, PH5CH8 cells were co-cultured with HCV-permissive, but RIG-I and TLR3-incompetent, Huh-7.5 cells infected with HJ3-5/GLuc2A, a reporter virus that expresses GLuc as a component of its polyprotein. Huh-7.5 cells were infected with virus for 6 hrs prior to being split and added to either MSR1-depleted PH5CH8/shMSR1 or control PH5CH8/shNT cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003345#ppat-1003345-g003" target="_blank">Fig. 3A</a>) at a ratio of 1∶2 to establish co-cultures. (<i>right</i>) <i>Gaussia</i> luciferase activity in supernatant fluids of HJ3-5/GLuc2A-infected co-cultures. Data shown are means ± s.d. from 3 replicate cultures and are representative of repeat, independent experiments. The difference in GLuc expression from shMSR1- vs. shNT-treated cells was significant by two-way ANOVA (p<0.0001). (<b>B</b>) (<i>left</i>) Co-cultures of PH5CH8/shMSRI (or control PH5CH8/shNT) cells and HJ3-5/GLuc2A virus-infected Huh-7.5 cells in which the cell types are separated by a semi-permeable membrane (Transwell system). (<i>right</i>) <i>Gaussia</i> luciferase activity in supernatant fluids of HJ3-5/GLuc2A virus-infected Huh-7.5 cells separated from the PH5CH8 cells by a semi-permeable membrane. Data shown are means ± s.d. from 3 replicate cultures. Unlike Huh-7.5 cells cultured in close continuity with PH5CH8 cells (panel A), there is no restriction to HCV replication when the cell types are separated by a permeable membrane. The difference in GLuc expression from shMSR1- vs. shNT-treated cells was not significant by two-way ANOVA (p = 0.20).</p

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

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    <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.</p

    Human macrophage scavenger receptor class A is required for TLR3 signaling triggered by HCV in hepatocytes.

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    <p>(<b>A</b>) Immunoblot analysis of MSR1 expression in (top panels) Huh7.5-TLR3 and (bottom panels) PH5CH8 cells transduced with MSR1-specific (shMSR1) or non-targeting control shRNA (shNT) lentiviruses. (<b>B</b>) Effect of shRNA-mediated MSR1 depletion on uptake of FITC-labeled poly(I:C) by Huh7.5-TLR3 or PH5CH8 cells. Cells were incubated with 10 µg/ml FITC-labeled poly(I:C) for 8, 16 or 24 hrs before harvest. Cells were washed extensively before fixation and analyzed by flow cytometry. Uptake of FITC-labeled poly(I:C) is expressed as fold-change in mean fluorescence intensity (MFI) compared to mock-treated cells. Statistical comparisons between the MFI at 8 hrs vs. the time indicated were made by two-way ANOVA with Bonferroni correction for multiple comparisons: * p≤0.02, ** p≤0.001. (<b>C</b>) Effect of shRNA-mediated MSR1 depletion on TLR3 signaling. IFN-β and PRD-II promoter activation was induced by extracellular poly(I:C) in MSR1-depleted (left) Huh7.5-TLR3 and (right) PH5CH8 cells exposed to 50 µg/ml of high molecular weight poly(I:C) for 6 hrs. Statistical comparisons in this and other panels in this figure were between the shNT and shMSR1-treated cells: * p≤0.02, ** p≤0.001. (<b>D</b>) Quantitative RT-PCR analysis of ISG56 mRNA in MSR1-depleted cells treated with poly(I:C) as in panel C. (<b>E</b>) Immunoblots of ISG15 expression in MSR1-depleted (left) Huh7.5-TLR3 and (right) PH5CH8 cells treated with poly(I:C) for 24 hrs. (<b>F</b>) Immunoblot of ISG15 in MSR1-depleted Huh7.5-TLR3 cells exposed to synthetic HCV dsRNA 50 µg/ml for 24 hrs. (<b>G</b>) Quantitative RT-PCR analysis of ISG56 mRNA in Huh7.5-TLR3/shMSR1 or Huh7.5-TLR3/shNT cells infected with HJ3-5 virus at an m.o.i. of 1 for 72 hrs. (<b>H</b>) Viral replication assessed by the fold-change in secreted <i>Gaussia</i> luciferase in cultures of Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells following infection with HJ3-5/GLuc2A virus. The level of <i>Gaussia</i> luciferase activity at each time point was calculated relative to that at 6 hrs after infection, which was set at 1. Data are the mean ± s.d. from three independent experiments. The difference in GLuc expression by Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells was significant by two-way ANOVA (p = 0.006). Bonferroni post-tests were used to estimate the significance of differences at individual time points: * p≤0.02. (<b>I</b>) Quantitative RT-PCR analysis of HCV RNA in Huh7.5-TLR3/shMSR1 vs. Huh7.5-TLR3/shNT cells 72 hrs after infection with HCV. RNA abundance in the Huh7.5-TLR3/shMSR1 was calculated relative to that in Huh7.5-TLR3/shNT, which was set at 100%. (<b>J</b>) Co-immunoprecipitation of HCV RNA with Flag-TLR3 in lysates of Huh7.5-TLR3/shMSR1 (lanes 3, 4, and 6) and Huh7.5-TLR3/shNT cells (lanes 1, 2 and 5) infected with HJ3-5 virus 72 hrs previously. Immunoprecipitation was with anti-Flag. 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 additional details.</p

    Reconstitution of MSR1 expression in MSR1-depleted cells restores TLR3 signaling triggered by HCV.

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    <p>(<b>A</b>) Reconstitution of MSR1 expression in Huh7.5-TLR3/shMSR1 cells. Myc-tagged MSR1 was stably expressed by retroviral transfer of a shMSR1-resistant vector (pCX4bsr/Myc-MSR1). Lysates were immunoprecipitated with anti-Myc, then subjected to immunoblotting as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003345#s4" target="_blank">Materials and Methods</a>. “Vector” = cells transduced with empty vector. (<b>B</b>) Restoration of poly(I:C) induction of IFN-β promoter activity by ectopically expressed Myc-MSR1 in Huh7.5-TLR3/shMSR1 cells. Cells were co-transfected with pCX4bsr/Myc-MSR1 (vs. empty vector), pIFN-β-Luc and pRL-CMV (internal control reporter) and cultured for 24 h, then treated with poly(I:C) (50 µg/ml) for 6 hrs prior to lysis and luciferase assay. Huh7.5-TLR3/shNT cells, transfected with empty vector, were included as a positive control. (<b>C</b>) Quantitative RT-PCR analysis of HCV RNA in Huh7.5-TLR3/shMSR1 cells stably expressing Myc-MSR1 (vs. empty vector) 72 hrs after infection with HJ3-5 virus at an m.o.i. of 1. (<b>D</b>) Co-immunoprecipitation of HCV RNA with Myc-MSR1 in lysates of Huh7.5-TLR3/shMSR1 cells stably expressing Myc-MSR1 (lanes 3 and 6) vs. empty vector (lanes 2 and 5). Cells were infected with HJ3-5 virus (m.o.i. = 1) 72 hrs prior to lysis. Immunoprecipitation was with anti-Myc, demonstrating association of HCV RNA with Myc-MSR1. 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.</p

    Scavenger receptor class A family transcript profiling of human hepatocyte-derived cell lines.

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    <p>(<b>A</b>) RT-PCR detection of class A scavenger receptor mRNAs in total cellular RNAs extracted from PH5CH8 cells transduced to express (“NT”) a non-targeting shRNA (PH5CH8/shNT cells), or (“T”) MSR1-specific shRNA (PH5CH8/shMSR1 cells). The parental PH5CH8 cells are T-antigen transformed normal adult human hepatocytes. Primers specific for MSR1 (SCARA1) transcript variant 1 (MSR1, SR-AI, or CD204) and transcript variant 2 (SR-AII), MARCO (SCARA2), SCARA3 (transcript variants 1 and 2), SCARA4, and SCARA5 (a putative scavenger receptor class A family member) were identical to those used by DeWitte-Orr et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003345#ppat.1003345-DeWitteOrr1" target="_blank">[22]</a>. PCR was carried through 35 cycles prior to separation of products on an agarose gel. (<b>B</b>) Similar class A scavenger receptor transcript profiling of Huh-7.5 cells derived from a human hepatocellular carcinoma, and 293FT cells (Invitrogen). *MSR1 has two transcript variants: variant 1 is commonly referred to as “MSR1”, as we refer to it here, but is otherwise known at SCARA1 or SR-AI. Transcript variant 2 is referred to as SR-AII. See text for additional details. **MARCO, macrophage receptor with collagenous structure, is otherwise known as SCARA2.</p

    Kinetics of HCV inhibition in infected cell culture following addition of an NS5A inhibitor, monitored using different GLuc reporter viruses.

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    <p>(A) Inhibition of the gt1a LPO<sup>S</sup> virus H77S.3/GLuc2A by elbasvir. (B) Inhibition of the gt2a LPO<sup>R</sup> virus JFH-1/QL/GLuc2A by elbasvir. (C) Maximum % inhibition (E<sub>max</sub>) of GLuc activity at different time points after addition of elbasvir to Huh7.5 cells infected with different cell culture infectious HCV genomes. LPO<sup>S</sup> viruses H77S.3/GLuc2A (gt1a) and N.2/GLuc2A (gt1b) are plotted in red. LPO<sup>R</sup> viruses H77D/GLuc2A (gt1a), JFH-1/QL/GLuc2A (gt2a) or HJ3-5/GLuc2A (gt1a/2a chimera). (D) Inhibition of the gt1a LPO<sup>R</sup> virus H77D-GLuc2A by elbasvir. (E) Fitness of different virus genomes used in (C) at 72 h post electroporation. Data shown represents GLuc activity relative to GLuc activity at 6 hours post electroporation to normalize for transfection efficiency. The replication incompetent reporter viruses H77S/AAG/GLuc2A and JFH-1/GND/GLuc2A contain point mutations in the NS5B polymerase and are included as mock-transfection controls.</p

    Results of the mathematical model fitted to data measuring the decline of GLuc-reporter activity from the H77S.3 or H77D variants under 10nM elbasvir treatment.

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    <p>Data and simulation using best fit parameter values are shown as ‘x’s and lines, respectively. The data and simulation shown here represent the 10nM elbasvir treatment from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006343#ppat.1006343.s005" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006343#ppat.1006343.s008" target="_blank">S8</a> Figs. Here, they are overlaid on the same graph to highlight the transient increase in GLuc activity observed at 4-8h following NS5A inhibitor addition to cell cultures infected with H77S.3/GLuc2A but not H77D/GLuc2A.</p
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