title: Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.
abstract: Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion.
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address: wsaw@dental.upenn.edu.
authors.forename: Wan Ting
authors.initials: WT
authors.lastname: Saw
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan; China-Japan Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, PR China. Electronic address: zmatsuda@ims.u-tokyo.ac.jp.
authors.forename: Zene
authors.initials: Z
authors.lastname: Matsuda
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address: roselyn@dental.upenn.edu.
authors.forename: Roselyn J
authors.initials: RJ
authors.lastname: Eisenberg
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address: ghc@dental.upenn.edu.
authors.forename: Gary H
authors.initials: GH
authors.lastname: Cohen
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address: doinaa2@dental.upenn.edu.
authors.forename: Doina
authors.initials: D
authors.lastname: Atanasiu
mesh_terms: D002459:Cell Fusion; D002478:Cells, Cultured; D017930:Genes, Reporter; D054884:Host-Pathogen Interactions; D007700:Kinetics; D008156:Luciferases / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D009154:Mutation; D018139:Simplexvirus / Q000502:physiology; D014760:Viral Fusion Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
chemical_list: D014760:Viral Fusion Proteins; D008156:Luciferases
keywords: Cell–cell fusion; Herpes simplex virus; Kinetics of fusion; Split luciferase assay