title: Establishment of an indicator cell system for hepatitis C virus.
abstract: Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.
authors.affiliation: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Mori
authors.forename: Hideki
authors.initials: H
authors.lastname: Tani
authors.forename: Takayuki
authors.initials: T
authors.lastname: Abe
authors.forename: Kohji
authors.initials: K
authors.lastname: Moriishi
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Kojima
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Okabe
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Masashi
authors.initials: M
authors.lastname: Tatsumi
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Matsuura
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014773:Virology / Q000379:methods; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C505371:MAVS protein, human; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus