Structural Determinants of Rotavirus Subgroup Specificity Mapped by Cryo-electron Microscopy

• Published in: Journal of molecular biology Vol. 356; no. 1; pp. 209 - 221
• Main Authors: Greig, Sarah L., Berriman, John A., O'Brien, Judith A., Taylor, John A., Bellamy, A. Richard, Yeager, Mark J., Mitra, Alok K.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 10.02.2006
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibodies, Viral - immunology; Binding Sites; capsid structure; Cattle; cryo-electron microscopy; Cryoelectron Microscopy; Epitopes - immunology; Fab fragments; Humans; image analysis; Models, Molecular; Protein Binding; Protein Structure, Quaternary; RNA-Directed DNA Polymerase - metabolism; Rotavirus; Rotavirus - chemistry; Rotavirus - classification; Rotavirus - ultrastructure; subgroup specificity; transcriptase; Viral Proteins - chemistry; Viral Proteins - classification; Viral Proteins - ultrastructure; Virion - chemistry; Virion - ultrastructure; capsid structure; DLP; transcriptase; image analysis; mAb; BSA; TCA; rotavirus; TLP; Fab fragments; cryo-EM; cryo-electron microscopy; subgroup specificity
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.11.049

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 Å and 28 Å 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.