title: Generation of infectious hepatitis C virus in immortalized human hepatocytes.
abstract: Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of approximately 50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded approximately 4.5 x 10(4) to 1 x 10(5) focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.
authors.affiliation: Department of Pathology, Saint Louis University, 3635 Vista Avenue, St. Louis, Missouri 63110, USA.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kanda
authors.forename: Arnab
authors.initials: A
authors.lastname: Basu
authors.forename: Robert
authors.initials: R
authors.lastname: Steele
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Jan S
authors.initials: JS
authors.lastname: Ryerse
authors.forename: Ranjit
authors.initials: R
authors.lastname: Ray
authors.forename: Ratna B
authors.initials: RB
authors.lastname: Ray
mesh_terms: D016923:Cell Death; D002999:Clone Cells; D003470:Culture Media; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral / Q000235:genetics; D006046:Gold; D016174:Hepacivirus / Q000302:isolation & purification* / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology* / Q000821:virology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D012367:RNA, Viral / Q000235:genetics; D014771:Virion / Q000378:metabolism; D014779:Virus Replication
chemical_list: D003470:Culture Media; D012367:RNA, Viral; D006046:Gold