title: Effects on sialic acid recognition of amino acid mutations in the carbohydrate-binding cleft of the rotavirus spike protein.
abstract: The rotavirus spike protein VP4 mediates attachment to host cells and subsequent membrane penetration. The VP8(*) domain of VP4 forms the spike tips and is proposed to recognize host-cell surface glycans. For sialidase-sensitive rotaviruses such as rhesus (RRV), this recognition involves terminal sialic acids. We show here that the RRV VP8(*)(64-224) protein competes with RRV infection of host cells, demonstrating its relevance to infection. In addition, we observe that the amino acids revealed by X-ray crystallography to be in direct contact with the bound sialic acid derivative methyl alpha-D-N-acetylneuraminide, and that are highly conserved amongst sialidase-sensitive rotaviruses, are residues that are also important in interactions with host-cell carbohydrates. Residues Arg101 and Ser190 of the RRV VP8(*) carbohydrate-binding site were mutated to assess their importance for binding to the sialic acid derivative and their competition with RRV infection of host cells. The crystallographic structure of the Arg(101)Ala mutant crystallized in the presence of the sialic acid derivative was determined at 295 K to a resolution of 1.9 A. Our multidisciplinary study using X-ray crystallography, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and competitive virus infectivity assays to investigate RRV wild-type and mutant VP8(*) proteins has provided the first evidence that the carbohydrate-binding cavity in RRV VP8(*) is used for host-cell recognition, and this interaction is not only with the sialic acid portion but also with other parts of the glycan structure.
authors.affiliation: Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland 4222, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Kraschnefski
authors.forename: Andrea
authors.initials: A
authors.lastname: Bugarcic
authors.forename: Fiona E
authors.initials: FE
authors.lastname: Fleming
authors.forename: Xing
authors.initials: X
authors.lastname: Yu
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Coulson
authors.forename: Helen
authors.initials: H
authors.lastname: Blanchard
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001665:Binding Sites; D002151:Calorimetry; D002241:Carbohydrates / Q000737:chemistry; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012401:Rotavirus / Q000737:chemistry*; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D000596:Amino Acids; D002241:Carbohydrates; D008024:Ligands; D017361:Viral Nonstructural Proteins; D014867:Water; D019158:N-Acetylneuraminic Acid