title: Characterization of rotavirus RNAs that activate innate immune signaling through the RIG-I-like receptors.
abstract: In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5' phosphate groups. RNAs lacking 2'-O-methylated 5' cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5' capping or 2'-O-methylation of the 5' cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.
authors.affiliation: Departments of Medicine and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Dina
authors.initials: D
authors.lastname: Uzri
authors.forename: Harry B
authors.initials: HB
authors.lastname: Greenberg
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007113:Immunity, Innate; D007370:Interferon Type I / Q000276:immunology; D008780:Methyltransferases / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D009713:Nucleotidyltransferases / Q000235:genetics; D012367:RNA, Viral / Q000276:immunology* / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics* / Q000276:immunology; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D014771:Virion / Q000235:genetics / Q000276:immunology
chemical_list: D036022:Capsid Proteins; D007370:Interferon Type I; D012367:RNA, Viral; C442546:VP3 protein, Rotavirus; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D053487:DEAD-box RNA Helicases
references.citation: PLoS Pathog. 2009 Jan;5(1):e1000280
references.pmid: 19180189
references.citation: Nat Immunol. 2011 Feb;12(2):137-43
references.pmid: 21217758
references.citation: Nature. 2010 Nov 18;468(7322):452-6
references.pmid: 21085181
references.citation: J Gen Virol. 2007 Feb;88(Pt 2):613-620
references.pmid: 17251580
references.citation: Arch Virol Suppl. 1996;12:141-52
references.pmid: 9015111
references.citation: Nat Immunol. 2004 Jul;5(7):730-7
references.pmid: 15208624
references.citation: J Infect Dis. 1984 May;149(5):694-702
references.pmid: 6202807
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4114-9
references.pmid: 15741273
references.citation: Curr Opin Virol. 2011 Dec;1(6):519-25
references.pmid: 22328912
references.citation: J Virol. 2004 Jul;78(14):7763-74
references.pmid: 15220450
references.citation: J Virol. 2011 Mar;85(5):1970-9
references.pmid: 21177809
references.citation: J Virol. 2009 May;83(9):4174-84
references.pmid: 19224987
references.citation: Wiley Interdiscip Rev RNA. 2010 Jul-Aug;1(1):152-72
references.pmid: 21956912
references.citation: J Virol. 2009 Oct;83(20):10322-35
references.pmid: 19656876
references.citation: J Biol Chem. 2009 Apr 10;284(15):9700-12
references.pmid: 19211564
references.citation: Proc Natl Acad Sci U S A. 1999 May 11;96(10):5428-33
references.pmid: 10318900
references.citation: J Virol. 2008 Aug;82(15):7578-90
references.pmid: 18495762
references.citation: J Virol. 2009 Jul;83(14):6987-94
references.pmid: 19420080
references.citation: J Virol. 2008 Jan;82(1):335-45
references.pmid: 17942531
references.citation: Nature. 2007 Apr 19;446(7138):916-920
references.pmid: 17392790
references.citation: Emerg Infect Dis. 2003 May;9(5):565-72
references.pmid: 12737740
references.citation: J Biol Chem. 1976 Aug 25;251(16):5043-53
references.pmid: 821947
references.citation: J Clin Microbiol. 1990 Jun;28(6):1342-7
references.pmid: 2166081
references.citation: J Gen Virol. 1976 May;31(2):245-50
references.pmid: 180243
references.citation: J Immunol. 2011 Feb 1;186(3):1618-26
references.pmid: 21187438
references.citation: Immunity. 2005 Jul;23(1):19-28
references.pmid: 16039576
references.citation: Mol Cell. 2005 Sep 16;19(6):727-40
references.pmid: 16153868
references.citation: J Gen Virol. 2008 Jan;89(Pt 1):1-47
references.pmid: 18089727
references.citation: J Gen Virol. 1977 Sep;36(3):395-402
references.pmid: 21225
references.citation: J Exp Med. 2008 Jul 7;205(7):1601-10
references.pmid: 18591409
references.citation: Immunity. 2009 Jul 17;31(1):25-34
references.pmid: 19576794
references.citation: Proc Natl Acad Sci U S A. 2006 May 30;103(22):8459-64
references.pmid: 16714379
references.citation: J Virol. 1993 May;67(5):2448-55
references.pmid: 8386262
references.citation: Pediatr Infect Dis J. 2011 Jan;30(1 Suppl):S1-5
references.pmid: 21183833
references.citation: Nat Biotechnol. 2006 May;24(5):559-65
references.pmid: 16648842
references.citation: Proc Natl Acad Sci U S A. 2009 Jul 21;106(29):12067-72
references.pmid: 19574455
references.citation: Nucleic Acids Res. 2010 Nov;38(21):7718-35
references.pmid: 20671030
references.citation: J Virol. 1983 Jul;47(1):125-36
references.pmid: 6306269
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):582-7
references.pmid: 17190814
references.citation: Cell. 2005 Sep 9;122(5):669-82
references.pmid: 16125763
references.citation: PLoS Pathog. 2013 Jan;9(1):e1003064
references.pmid: 23359266
references.citation: J Biol Chem. 1981 Jun 25;256(12):6507-14
references.pmid: 7240222
references.citation: J Virol. 1987 Nov;61(11):3479-84
references.pmid: 2822955
references.citation: J Immunol. 2005 Sep 1;175(5):2851-8
references.pmid: 16116171
references.citation: Cell. 2010 Feb 5;140(3):397-408
references.pmid: 20144762
references.citation: Nature. 2008 Jul 24;454(7203):523-7
references.pmid: 18548002
references.citation: PLoS Pathog. 2012;8(5):e1002670
references.pmid: 22570612
references.citation: J Virol. 2011 Apr;85(8):3717-32
references.pmid: 21307186
references.citation: J Virol. 2002 Dec;76(24):13001-14
references.pmid: 12438626
references.citation: PLoS Pathog. 2010 Jun 03;6(6):e1000931
references.pmid: 20532161
references.citation: J Virol. 2002 Jan;76(2):560-8
references.pmid: 11752147
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16743-8
references.pmid: 18948594
references.citation: J Virol. 1998 Sep;72(9):7387-96
references.pmid: 9696835
references.citation: PLoS One. 2007 Mar 14;2(3):e279
references.pmid: 17356690
references.citation: J Virol. 2013 Aug;87(15):8307-16
references.pmid: 23698306
references.citation: Mol Cell. 2008 Feb 1;29(2):169-79
references.pmid: 18243112
references.citation: J Gen Virol. 2013 Jun;94(Pt 6):1151-1160
references.pmid: 23486667
references.citation: Mol Cell. 2008 Feb 29;29(4):428-40
references.pmid: 18242112
references.citation: Nat Immunol. 2005 Oct;6(10):981-8
references.pmid: 16127453
references.citation: Science. 2006 Nov 10;314(5801):997-1001
references.pmid: 17038589
references.citation: Immunity. 2006 May;24(5):633-42
references.pmid: 16713980
references.citation: Cancer Res. 1982 Sep;42(9):3858-63
references.pmid: 6286115
references.citation: Nucleic Acids Res. 1991 Jun 11;19(11):2993-3000
references.pmid: 2057357
references.citation: Virol J. 2011 Dec 08;8:526
references.pmid: 22152002
references.citation: Viruses. 2011 Jun;3(6):920-40
references.pmid: 21994762
references.citation: J Virol. 2009 Oct;83(20):10761-9
references.pmid: 19656871
references.citation: J Virol. 2007 Mar;81(5):2179-86
references.pmid: 17151117
references.citation: Science. 2006 Nov 10;314(5801):994-7
references.pmid: 17038590
references.citation: Lancet Infect Dis. 2012 Feb;12(2):136-41
references.pmid: 22030330
references.citation: RNA. 2006 Jan;12(1):133-46
references.pmid: 16301600
references.citation: J Virol. 2010 Oct;84(20):10457-66
references.pmid: 20631127
references.citation: J Clin Microbiol. 1990 Jun;28(6):1198-203
references.pmid: 2166076
references.citation: Nature. 2007 Aug 16;448(7155):816-9
references.pmid: 17653195
references.citation: Nature. 2005 Oct 20;437(7062):1167-72
references.pmid: 16177806
references.citation: Virology. 1995 May 10;209(1):274-80
references.pmid: 7747480
references.citation: J Virol. 2009 May;83(10):4942-51
references.pmid: 19244315
references.citation: J Virol. 2007 May;81(9):4473-81
references.pmid: 17301153