title: Development and validation of a high-throughput screening assay for the hepatitis C virus p7 viroporin.
abstract: The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.
authors.affiliation: Department of Biological Sciences, Research and Development, Boehringer Ingelheim (Canada) Ltd., Laval, QC, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Gervais
authors.forename: Florence
authors.initials: F
authors.lastname: Dô
authors.forename: Ariane
authors.initials: A
authors.lastname: Cantin
authors.forename: George
authors.initials: G
authors.lastname: Kukolj
authors.forename: Peter W
authors.initials: PW
authors.lastname: White
authors.forename: Annick
authors.initials: A
authors.lastname: Gauthier
authors.forename: Frédéric H
authors.initials: FH
authors.lastname: Vaillancourt
mesh_terms: D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008555:Melitten / Q000378:metabolism; D010539:Permeability; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D007473:Ion Channels; D008081:Liposomes; D008555:Melitten; D010743:Phospholipids; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins