title: The Molecular Chaperone GRP78 Contributes to Toll-like Receptor 3-mediated Innate Immune Response to Hepatitis C Virus in Hepatocytes.
abstract: Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-κB and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferon-stimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3-dependent innate immune response to HCV in hepatocytes.
authors.affiliation: From the Departments of Microbiology, Immunology and Biochemistry and.
authors.forename: Dahai
authors.initials: D
authors.lastname: Wei
authors.affiliation: From the Departments of Microbiology, Immunology and Biochemistry and.
authors.forename: Nan L
authors.initials: NL
authors.lastname: Li
authors.affiliation: the Department of Infectious Diseases, Henan Provincial People's Hospital, Zhengzhou, Henan 450003, China.
authors.forename: Yanli
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: From the Departments of Microbiology, Immunology and Biochemistry and.
authors.forename: Baoming
authors.initials: B
authors.lastname: Liu
authors.affiliation: From the Departments of Microbiology, Immunology and Biochemistry and.
authors.forename: Kattareeya
authors.initials: K
authors.lastname: Kumthip
authors.affiliation: the SRI International, Harrisonburg, Virginia 22802, and.
authors.forename: Tony T
authors.initials: TT
authors.lastname: Wang
authors.affiliation: the Department of Public Health Sciences, University of Chicago, Chicago, Illinois 60637.
authors.forename: Dezheng
authors.initials: D
authors.lastname: Huo
authors.affiliation: Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Jesse F
authors.initials: JF
authors.lastname: Ingels
authors.affiliation: Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Lu
authors.initials: L
authors.lastname: Lu
authors.affiliation: the Department of Infectious Diseases, Henan Provincial People's Hospital, Zhengzhou, Henan 450003, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Shang
authors.affiliation: From the Departments of Microbiology, Immunology and Biochemistry and kli1@uthsc.edu.
authors.forename: Kui
authors.initials: K
authors.lastname: Li
mesh_terms: D000328:Adult; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D011992:Endosomes / Q000378:metabolism / Q000821:virology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D016174:Hepacivirus / Q000276:immunology* / Q000502:physiology; D019698:Hepatitis C, Chronic / Q000235:genetics / Q000378:metabolism / Q000821:virology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000821:virology; D008297:Male; D018613:Microscopy, Confocal; D008875:Middle Aged; D011070:Poly I-C / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051196:Toll-Like Receptor 3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D018946:Chemokine CCL5; D054357:Chemokine CXCL10; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D011070:Poly I-C
keywords: GRP78; Hepatitis C virus (HCV); RANTES; Toll-like receptor-3; chemokine; hepatocyte; innate immunity; interferon regulatory factor-3; interferon stimulated gene; molecular chaperone