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Virology. 2015 Mar;477:32-41. doi: 10.1016/j.virol.2015.01.003. Epub 2015 Jan 28.

Electron microscopic analysis of rotavirus assembly-replication intermediates.

Author information

1
Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA, USA.
2
Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA, USA; Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA. Electronic address: mcdonaldsa@vtc.vt.edu.

Abstract

Rotaviruses (RVs) replicate their segmented, double-stranded RNA genomes in tandem with early virion assembly. In this study, we sought to gain insight into the ultrastructure of RV assembly-replication intermediates (RIs) using transmission electron microscopy (EM). Specifically, we examined a replicase-competent, subcellular fraction that contains all known RV RIs. Three never-before-seen complexes were visualized in this fraction. Using in vitro reconstitution, we showed that ~15-nm doughnut-shaped proteins in strings were nonstructural protein 2 (NSP2) bound to viral RNA transcripts. Moreover, using immunoaffinity-capture EM, we revealed that ~20-nm pebble-shaped complexes contain the viral RNA polymerase (VP1) and RNA capping enzyme (VP3). Finally, using a gel purification method, we demonstrated that ~30-70-nm electron-dense, particle-shaped complexes represent replicase-competent core RIs, containing VP1, VP3, and NSP2 as well as capsid proteins VP2 and VP6. The results of this study raise new questions about the interactions among viral proteins and RNA during the concerted assembly-replicase process.

KEYWORDS:

Assembly; Electron microscopy; Genome replication; Intermediates; Replicase; Rotavirus; Subviral particles; Ultrastructure

PMID:
25635339
PMCID:
PMC4359669
DOI:
10.1016/j.virol.2015.01.003
[Indexed for MEDLINE]
Free PMC Article

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