title: A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells.
abstract: As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K(k)] through the (Gal4)(5)-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K(k) as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.
authors.affiliation: Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Adrien
authors.initials: A
authors.lastname: Breiman
authors.affiliation: Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Vitour
authors.affiliation: Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Myriam
authors.initials: M
authors.lastname: Vilasco
authors.affiliation: Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Ottone
authors.affiliation: INSERM, U632, F-34293 Montpellier, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Molina
authors.affiliation: INSERM, U632, F-34293 Montpellier, France.
authors.forename: Lydiane
authors.initials: L
authors.lastname: Pichard
authors.affiliation: Etablissement Français du Sang (EFS) Pyrénées-Méditerranée, F-34094 Montpellier, France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Fournier
authors.affiliation: Institut Pasteur de Lille, Groupe Hépatite C, Institut de Biologie de Lille, F-59021 Lille, France.
authors.forename: David
authors.initials: D
authors.lastname: Delgrange
authors.affiliation: Laboratoire Virologie Moléculaire et Vectorologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Charneau
authors.affiliation: CHU Amiens, Laboratoire de Virologie, 80054 Amiens, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.affiliation: Institut Pasteur de Lille, Groupe Hépatite C, Institut de Biologie de Lille, F-59021 Lille, France.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.affiliation: INSERM, U632, F-34293 Montpellier, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Maurel
authors.affiliation: Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
authors.forename: Eliane F
authors.initials: EF
authors.lastname: Meurs
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000737:chemistry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006183:H-2 Antigens / Q000096:biosynthesis; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D002352:Carrier Proteins; D015500:Chloramphenicol O-Acetyltransferase; D049452:Green Fluorescent Proteins; D006183:H-2 Antigens; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C451899:ERP44 protein, human; C070633:Gal-VP16; C024755:H-2K(K) antigen; 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; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase