title: Binding dynamics of hepatitis C virus' NS5A amphipathic peptide to cell and model membranes.
abstract: Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.
authors.affiliation: Department of Materials Science and Engineering, Stanford University, 381 North-South Mall, Stauffer III, Stanford, CA 94305, USA.
authors.forename: Nam-Joon
authors.initials: NJ
authors.lastname: Cho
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Cheong
authors.forename: ChoongHo
authors.initials: C
authors.lastname: Lee
authors.forename: Curtis W
authors.initials: CW
authors.lastname: Frank
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Glenn
mesh_terms: D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D049452:Green Fluorescent Proteins; D008051:Lipid Bilayers; D010447:Peptide Hydrolases; D010455:Peptides; D014357:Trypsin; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase