title: Structural Insights into Reovirus σ1 Interactions with Two Neutralizing Antibodies.
abstract: Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus.
  
  
  
  Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
authors.forename: Melanie H
authors.initials: MH
authors.lastname: Dietrich
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.|Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.|Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Ogden
authors.affiliation: Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.|Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Sarah P
authors.initials: SP
authors.lastname: Katen
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Reiss
authors.affiliation: Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.|Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Danica M
authors.initials: DM
authors.lastname: Sutherland
authors.affiliation: Vanderbilt Antibody and Protein Resource, Vanderbilt Institute for Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.|Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Carnahan
authors.affiliation: Vanderbilt Antibody and Protein Resource, Vanderbilt Institute for Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Goff
authors.affiliation: Vanderbilt Antibody and Protein Resource, Vanderbilt Institute for Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors.forename: Tracy
authors.initials: T
authors.lastname: Cooper
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA terence.dermody@chp.edu thilo.stehle@uni-tuebingen.de.|Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University Medical Center, Nashville, Tennessee, USA.|Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany terence.dermody@chp.edu thilo.stehle@uni-tuebingen.de.|Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: Thilo
authors.initials: T
authors.lastname: Stehle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D000914:Antibodies, Viral / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D051379:Mice; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009500:Neutralization Tests; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D012087:Reoviridae / Q000187:drug effects / Q000502:physiology; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007140:Immunoglobulin Fab Fragments; D014764:Viral Proteins
keywords: Fab fragments; crystal structure; flow cytometry; immune escape; mammalian reoviruses; monoclonal antibodies; quaternary binding epitope; receptor binding; surface plasmon resonance; virus neutralization