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A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells

Adrien Breiman^1,

¹ Unité Hépacivirus, Département de Virologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France
² INSERM, U632, F-34293 Montpellier, France
³ Etablissement Français du Sang (EFS) Pyrénées-Méditerranée, F-34094 Montpellier, France
⁴ Institut Pasteur de Lille, Groupe Hépatite C, Institut de Biologie de Lille, F-59021 Lille, France
⁵ Laboratoire Virologie Moléculaire et Vectorologie, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France
⁶ CHU Amiens, Laboratoire de Virologie, 80054 Amiens, France

Correspondence
Eliane F. Meurs
emeurs{at}pasteur.fr

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.

A supplementary table showing all oligonucleotides used for the construction of plasmids is available in JGV Online.

Present address: Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.