title: Influenza virus-membrane fusion triggered by proton uncaging for single particle studies of fusion kinetics.
abstract: We report a method for studying membrane fusion, focusing on influenza virus fusion to lipid bilayers, which provides high temporal resolution through the rapid and coordinated initiation of individual virus fusion events. Each fusion event proceeds through a series of steps, much like multistep chemical reaction. Fusion is initiated by a rapid decrease in pH that accompanies the "uncaging" of an effector molecule from o-nitrobenzaldehyde, a photoisomerizable compound that releases a proton to the surrounding solution within microseconds of long-wave ultraviolet irradiation. In order to quantify pH values upon UV irradiation and uncaging, we introduce a simple silica nanoparticle pH sensor, useful for reporting the pH in homogeneous nanoliter volumes under conditions where traditional organic dye-type pH probes fail. Subsequent single-virion fusion events are monitored using total internal reflection fluorescence microscopy. Statistical analysis of these stochastic events uncovers kinetic information about the fusion reaction. This approach reveals that the kinetic parameters obtained from the data are sensitive to the rate at which protons are delivered to the bound viruses. Higher resolution measurements can enhance fundamental fusion studies and aid antiviral antifusogenic drug development.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States.
authors.forename: Deirdre A
authors.initials: DA
authors.lastname: Costello
authors.forename: Donald W
authors.initials: DW
authors.lastname: Lee
authors.forename: Jennifer
authors.initials: J
authors.lastname: Drewes
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Vasquez
authors.forename: Kassandra
authors.initials: K
authors.lastname: Kisler
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wiesner
authors.forename: Lois
authors.initials: L
authors.lastname: Pollack
authors.forename: Gary R
authors.initials: GR
authors.lastname: Whittaker
authors.forename: Susan
authors.initials: S
authors.lastname: Daniel
mesh_terms: D002462:Cell Membrane / Q000821:virology*; D054884:Host-Pathogen Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053122:Influenza A Virus, H3N2 Subtype / Q000502:physiology*; D007251:Influenza, Human / Q000821:virology*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D053758:Nanoparticles / Q000032:analysis; D011522:Protons; D012822:Silicon Dioxide / Q000032:analysis; D053586:Virus Internalization
chemical_list: D008051:Lipid Bilayers; D011522:Protons; D012822:Silicon Dioxide