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.
authors: Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA, USA.
authors: Crystal E
authors: CE
authors: Boudreaux
authors: Virginia Tech Carilion School of Medicine and Research Institute, Roanoke, VA, USA.
authors: Deborah F
authors: DF
authors: Kelly
authors: 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.
authors: Sarah M
authors: SM
authors: McDonald
mesh_terms: D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D000882:Haplorhini; D046911:Macromolecular Substances / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism; D012401:Rotavirus / Q000502:physiology* / Q000648:ultrastructure*; D014764:Viral Proteins / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D046911:Macromolecular Substances; D012367:RNA, Viral; D014764:Viral Proteins
keywords: Assembly; Electron microscopy; Genome replication; Intermediates; Replicase; Rotavirus; Subviral particles; Ultrastructure
references: J Struct Biol. 1996 Jan-Feb;116(1):190-9
references: 8742743
references: Nat Struct Biol. 1997 Feb;4(2):118-21
references: 9033591
references: J Gen Virol. 1999 Feb;80 ( Pt 2):333-9
references: 10073692
references: J Virol. 2005 Dec;79(24):15165-74
references: 16306588
references: Curr Top Microbiol Immunol. 2006;309:169-87
references: 16909900
references: J Mol Biol. 2006 Sep 22;362(3):539-54
references: 16934294
references: J Virol. 2006 Nov;80(21):10829-35
references: 16928740
references: J Virol. 1999 Dec;73(12):9934-43
references: 10559306
references: J Virol. 2001 May;75(10):4519-27
references: 11312322
references: EMBO J. 2002 Mar 1;21(5):885-97
references: 11867517
references: Nature. 2002 May 16;417(6886):311-5
references: 12015608
references: J Gen Virol. 2004 Mar;85(Pt 3):625-34
references: 14993647
references: J Biol Chem. 2004 Mar 12;279(11):10624-33
references: 14699117
references: Virus Res. 2004 Apr;101(1):67-81
references: 15010218
references: J Gen Virol. 1978 May;39(2):205-17
references: 206653
references: J Gen Virol. 1980 Jan;46(1):75-85
references: 6243348
references: Arch Virol. 1981;70(1):33-41
references: 6277282
references: J Virol. 1982 Sep;43(3):1113-7
references: 6292454
references: J Virol. 1984 Mar;49(3):665-73
references: 6321755
references: Virology. 1986 Dec;155(2):655-65
references: 3024405
references: Virus Res. 1986 Dec;6(3):217-33
references: 2437720
references: Virology. 1988 Oct;166(2):358-65
references: 2845649
references: Virology. 1989 Oct;172(2):616-27
references: 2552662
references: J Gen Virol. 1990 May;71 ( Pt 5):1087-94
references: 2161046
references: Virology. 1990 Sep;178(1):143-51
references: 2167546
references: Nature. 1996 Aug 1;382(6590):471-3
references: 8684490
references: Mem Inst Oswaldo Cruz. 2006 Sep;101(6):603-11
references: 17072471
references: Adv Virus Res. 2007;69:167-201
references: 17222694
references: J Virol. 2007 Nov;81(22):12272-84
references: 17804496
references: Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):1867-72
references: 18238898
references: Structure. 2008 Nov 12;16(11):1678-88
references: 19000820
references: J Virol. 2009 Feb;83(4):1754-66
references: 19036817
references: Nat Protoc. 2008;3(12):1941-74
references: 19180078
references: Science. 2009 Jun 12;324(5933):1444-7
references: 19520960
references: Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10644-8
references: 19487668
references: Curr Protoc Microbiol. 2009 Nov;Chapter 15:Unit 15C.3
references: 19885940
references: J Mol Biol. 2010 Mar 26;397(2):587-99
references: 20122940
references: J Biol Chem. 2010 Jun 11;285(24):18123-8
references: 20351108
references: EMBO J. 2011 Jan 19;30(2):408-16
references: 21157433
references: J Virol. 2011 Apr;85(7):3095-105
references: 21248043
references: Nat Rev Microbiol. 2012 Mar;10(3):165-77
references: 22266782
references: Curr Opin Virol. 2012 Aug;2(4):373-9
references: 22595300
references: J Virol. 2012 Oct;86(19):10547-57
references: 22811529
references: PLoS One. 2012;7(10):e47947
references: 23110139
references: Lab Chip. 2013 Jan 21;13(2):216-9
references: 23208001
references: J Mol Biol. 2013 Jan 9;425(1):124-32
references: 23089332
references: Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):13114-9
references: 23878220
references: Antiviral Res. 2013 Dec;100(3):615-35
references: 24129118
references: Virus Res. 2013 Dec 26;178(2):252-63
references: 24091366
references: J Virol. 2014 Jan;88(2):786-98
references: 24198401
references: PLoS Pathog. 2014 May;10(5):e1004157
references: 24873828
references: J Virol. 2014 Nov;88(21):12866-81
references: 25165107