title: Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system.
abstract: Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.
authors.affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Corine
authors.initials: C
authors.lastname: StGelais
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Tuthill
authors.forename: Dean S
authors.initials: DS
authors.lastname: Clarke
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
authors.forename: Stephen
authors.initials: S
authors.lastname: Griffin
mesh_terms: D000143:Acids; D000547:Amantadine / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D005452:Fluoresceins / Q000378:metabolism; D016174:Hepacivirus / Q000737:chemistry*; D007473:Ion Channels / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010539:Permeability; D018272:Porins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D000143:Acids; D000547:Amantadine; D000998:Antiviral Agents; D005452:Fluoresceins; D007473:Ion Channels; D008081:Liposomes; D018272:Porins; D011994:Recombinant Proteins; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins; C024098:6-carboxyfluorescein