A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells.
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.
Breiman
Adrien
A
Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
Vitour
Damien
D
Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
Vilasco
Myriam
M
Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
Ottone
Catherine
C
Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
Molina
Sonia
S
INSERM, U632, F-34293 Montpellier, France.
Pichard
Lydiane
L
INSERM, U632, F-34293 Montpellier, France.
Fournier
Chantal
C
Etablissement Français du Sang (EFS) Pyrénées-Méditerranée, F-34094 Montpellier, France.
Delgrange
David
D
Institut Pasteur de Lille, Groupe Hépatite C, Institut de Biologie de Lille, F-59021 Lille, France.
Charneau
Pierre
P
Laboratoire Virologie Moléculaire et Vectorologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
Duverlie
Gilles
G
CHU Amiens, Laboratoire de Virologie, 80054 Amiens, France.
Wychowski
Czeslaw
C
Institut Pasteur de Lille, Groupe Hépatite C, Institut de Biologie de Lille, F-59021 Lille, France.
Maurel
Patrick
P
INSERM, U632, F-34293 Montpellier, France.
Meurs
Eliane F
EF
Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France.
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
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