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Title: Electron microscopic analysis of rotavirus assembly-replication intermediates

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. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.

Authors:
;  [1];  [1];  [2]
  1. Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA (United States)
  2. (United States)
Publication Date:
OSTI Identifier:
22470156
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 477; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ELECTRONS; ENZYMES; GAIN; GELS; IMAGES; IN VITRO; PARTICLES; RNA; TRANSMISSION ELECTRON MICROSCOPY; VIRUSES

Citation Formats

Boudreaux, Crystal E., Kelly, Deborah F., McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu, and Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA. Electron microscopic analysis of rotavirus assembly-replication intermediates. United States: N. p., 2015. Web. doi:10.1016/J.VIROL.2015.01.003.
Boudreaux, Crystal E., Kelly, Deborah F., McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu, & Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA. Electron microscopic analysis of rotavirus assembly-replication intermediates. United States. doi:10.1016/J.VIROL.2015.01.003.
Boudreaux, Crystal E., Kelly, Deborah F., McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu, and Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA. Sun . "Electron microscopic analysis of rotavirus assembly-replication intermediates". United States. doi:10.1016/J.VIROL.2015.01.003.
@article{osti_22470156,
title = {Electron microscopic analysis of rotavirus assembly-replication intermediates},
author = {Boudreaux, Crystal E. and Kelly, Deborah F. and McDonald, Sarah M., E-mail: mcdonaldsa@vtc.vt.edu and Department of Biomedical Sciences and Pathobiology, Virginia—Maryland Regional College of Veterinary Medicine, Blacksburg, VA},
abstractNote = {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. - Highlights: • Rotaviruses replicate their genomes in tandem with early virion assembly. • Little is known about rotavirus assembly-replication intermediates. • Assembly-replication intermediates were imaged using electron microscopy.},
doi = {10.1016/J.VIROL.2015.01.003},
journal = {Virology},
number = ,
volume = 477,
place = {United States},
year = {Sun Mar 15 00:00:00 EDT 2015},
month = {Sun Mar 15 00:00:00 EDT 2015}
}