title: Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies.
abstract: Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, USA.
authors.forename: Brett D
authors.initials: BD
authors.lastname: Welch
authors.affiliation: Department of Biochemistry and Molecular Biology and the Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Marcin
authors.initials: M
authors.lastname: Paduch
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, USA.
authors.forename: George P
authors.initials: GP
authors.lastname: Leser
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Bergman
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Kors
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA.
authors.forename: Reay G
authors.initials: RG
authors.lastname: Paterson
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
authors.affiliation: Department of Biochemistry and Molecular Biology and the Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA koss@bsd.uchicago.edu ralamb@northwestern.edu.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA Howard Hughes Medical Institute, Northwestern University, Evanston, Illinois, USA koss@bsd.uchicago.edu ralamb@northwestern.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lamb
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008854:Microscopy, Electron; D010252:Paramyxoviridae / Q000502:physiology*; D014764:Viral Proteins / Q000276:immunology / Q000502:physiology*
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; D006023:Glycoproteins; D014764:Viral Proteins