title: Rotavirus VP6 modified for expression on the plasma membrane forms arrays and exhibits enhanced immunogenicity.
abstract: The major inner capsid protein of rotavirus is VP6, a 42-kDa polypeptide that forms the icosahedral surface of the rotavirus single-shelled particle. A chimeric form of VP6 (VP6sc) was constructed containing an upstream leader sequence derived from the influenza virus hemagglutinin and a downstream membrane-spanning (anchor) domain from a mouse immunoglobulin gene. When VP6sc was expressed in cells using a recombinant vaccinia virus, the protein was transported, glycosylated, and anchored in the plasma membrane as a trimer with the major domains of the protein orientated externally. Immunofluorescence and immunolabeling with colloidal gold indicated that VP6sc also localized in patches on the cell surface; electron microscopy revealed that the protein assembled into two-dimensional arrays which exhibited the same periodicity as the paracrystalline arrays formed by purified (viral) VP6. Mice inoculated with a recombinant vaccinia virus that expressed VP6sc produced rotavirus-specific antibodies at a titer 10 times higher than that achieved when wild-type, intracellular VP6 was delivered in the same way. Presentation at the cell surface therefore may represent a general method for enhancing the immunogenicity of rotavirus proteins.
authors.affiliation: Centre for Gene Technology, School of Biological Sciences, University of Auckland, New Zealand.
authors.forename: D A
authors.initials: DA
authors.lastname: Reddy
authors.forename: C C
authors.initials: CC
authors.lastname: Bergmann
authors.forename: J C
authors.initials: JC
authors.lastname: Meyer
authors.forename: J
authors.initials: J
authors.lastname: Berriman
authors.forename: G W
authors.initials: GW
authors.lastname: Both
authors.forename: B E
authors.initials: BE
authors.lastname: Coupar
authors.forename: D B
authors.initials: DB
authors.lastname: Boyle
authors.forename: M E
authors.initials: ME
authors.lastname: Andrew
authors.forename: A R
authors.initials: AR
authors.lastname: Bellamy
mesh_terms: D000818:Animals; D000956:Antigens, Viral; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012401:Rotavirus / Q000648:ultrastructure*
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C061332:VP6 protein, Rotavirus