34 research outputs found
MSR1-dependent TLR3 sensing of HCV infection in neighboring cells restricts viral replication.
<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 1âś2 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 Âą 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 Âą 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 (pâ=â0.20).</p
Poly(I:C) induced TLR3 signaling is dependent upon a conserved collagen superfamily domain in MSR1.
<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. âMacscavââ=âconserved macrophage scavenger receptor domain; âSRCRââ=âCys-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/Î321â339 (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 Âľg/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. âFL2-Hââ=âfluorescent 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.â=â1) 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), Î321â339 (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.
<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): * pâ¤0.02, ** pâ¤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 Âľg/ml of ssRNA or dsRNA for 6 hrs prior to extraction of RNA. Statistical comparisons were made with cultures not treated with RNA: * pâ¤0.02, ** pâ¤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.â=â1) 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
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. * pâ¤0.02. (<i>right</i>) qRT-PCR detection of TLR3-specific mRNA in cells transfected with the siTLR3 or siCont siRNAs. * pâ¤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 ÂľM, 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
Scavenger receptor class A family transcript profiling of human hepatocyte-derived cell lines.
<p>(<b>A</b>) RT-PCR detection of class A scavenger receptor mRNAs in total cellular RNAs extracted from PH5CH8 cells transduced to express (âNTâ) a non-targeting shRNA (PH5CH8/shNT cells), or (âTâ) 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 âMSR1â, 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
Human macrophage scavenger receptor class A is required for TLR3 signaling triggered by HCV in hepatocytes.
<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 Âľg/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: * pâ¤0.02, ** pâ¤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 Âľg/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: * pâ¤0.02, ** pâ¤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 Âľg/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 Âą 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 (pâ=â0.006). Bonferroni post-tests were used to estimate the significance of differences at individual time points: * pâ¤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.
<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>. âVectorââ=âcells 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 Âľg/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.â=â1) 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
microRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection
<div><p>Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.</p> </div
miR-122 expression in chimpanzee liver tissue.
<p>(<b>A</b>) Hepatic miR-122 abundance in liver biopsies from chimpanzees infected with HBV or HCV, or not infected with either virus (ânormalâ). Statistical significance was assessed by non-paired two-sided t test. Bars represent mean values. (<b>B</b>) Liver miR-122 expression plotted against serum HCV RNA abundance from acutely HCV-infected chimpanzees. r<sub>s</sub> = Spearman rank-order correlation coefficient.</p
miR-122 expression in paired HCC and non-tumor liver tissue from patients with chronic HBV and HCV infection and control, non-infected liver tissue.
<p>(<b>A</b>) miR-122 abundance quantified by qRT-PCR in paired tumor and non-tumor tissues and non-infected (ânormalâ) liver from patients undergoing resection of metastatic tumors, normalized to total RNA. (<b>B</b>) Relative miR-122 abundance normalized to miR-24 abundance in the same tissues. (<b>C</b>) miR-122 abundance in HCC classified histologically as âmoderately differentiatedâ, paired non-tumor tissue from the same patients, and non-infected (ânormalâ) liver. (<b>D</b>) miR-122 abundance in the subset of tissues shown in panel C, normalized to miR-24 abundance. The statistical significance of differences between paired observations was estimated using the paired t test, while differences between non-paired observations were analyzed by the Mann-Whitney test.</p