title: Structural determinants of rotavirus subgroup specificity mapped by cryo-electron microscopy.
abstract: The rotavirus double-layered particle (DLP) is a molecular machine that transcribes 11 genomic segments of double-stranded RNA into full-length mRNA segments during viral replication. DLPs from the human Wa strain of virus, belonging to subgroup II (SG II), possess a significantly reduced level of transcriptase activity compared to bovine UK DLPs that belong to subgroup I (SG I). Cryo-electron microscopy and icosahedral image analysis was used to define the structural basis for this difference in transcriptase activity and to derive three-dimensional density maps of bovine UK and human Wa DLPs at 26 angstroms and 28 angstroms resolution, respectively. The two rotavirus strains had the same diameter, T = 13 l icosahedral lattice symmetry and size of the VP6 trimers on the surface of the DLPs. However, the Wa particles displayed a remarkable absence of VP6 trimers surrounding each 5-fold vertex position. To further explore these structural differences, three-dimensional reconstructions were generated of DLPs decorated with Fab fragments derived from subgroup-specific monoclonal antibodies. The X-ray structures of VP6 and a generic Fab fragment were then docked into the cryo-electron microscopy density maps, which allowed us to propose at "pseudo-atomic" resolution the locations of the amino acid residues defining the subgroup-specific epitopes. Our results demonstrate a correlation between the structure of the VP6 layer and the transcriptase activity of the particles, and suggest that the stability of VP6 trimers, specifically those at the icosahedral 5-fold axes, may be critical for mRNA synthesis. Thus, subgroup specificity of rotavirus may reflect differences in the architecture of the double-layered particle, with resultant consequences for viral mRNA synthesis.
authors.affiliation: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Greig
authors.forename: John A
authors.initials: JA
authors.lastname: Berriman
authors.forename: Judith A
authors.initials: JA
authors.lastname: O'Brien
authors.forename: John A
authors.initials: JA
authors.lastname: Taylor
authors.forename: A Richard
authors.initials: AR
authors.lastname: Bellamy
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Yeager
authors.forename: Alok K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001665:Binding Sites; D002417:Cattle; D020285:Cryoelectron Microscopy; D000939:Epitopes / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry / Q000145:classification* / Q000648:ultrastructure*; D014764:Viral Proteins / Q000737:chemistry / Q000145:classification / Q000648:ultrastructure; D014771:Virion / Q000737:chemistry / Q000648:ultrastructure
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase