title: Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.
abstract: Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Gallagher
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: Doina
authors.initials: D
authors.lastname: Atanasiu
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: Wan Ting
authors.initials: WT
authors.lastname: Saw
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Paradisgarten
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: J Charles
authors.initials: JC
authors.lastname: Whitbeck
authors.affiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Roselyn J
authors.initials: RJ
authors.lastname: Eisenberg
authors.affiliation: Department of Microbiology, School of Dental Medicine, University of Pennsylvania Philadelphia, Pennsylvania, United States of America.
authors.forename: Gary H
authors.initials: GH
authors.lastname: Cohen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D006561:Herpes Simplex / Q000378:metabolism* / Q000821:virology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D018139:Simplexvirus / Q000502:physiology; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; C040495:glycoprotein B, Simplexvirus; C054562:yellow fluorescent protein, Bacteria