title: Prolonged activity of the pestiviral RNase Erns as an interferon antagonist after uptake by clathrin-mediated endocytosis.
abstract: The RNase activity of the envelope glycoprotein E(rns) of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E(rns) is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E(rns). Here, we show that the activity of soluble E(rns) as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E(rns) protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E(rns) mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E(rns) remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E(rns) acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance.
  
  
  OBJECTIVE
  The pestiviral RNase E(rns) was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E(rns) at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E(rns) is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E(rns) remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.
authors.affiliation: Institute of Veterinary Virology, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Zürcher
authors.affiliation: Institute of Veterinary Virology, University of Bern, Bern, Switzerland.
authors.forename: Kay-Sara
authors.initials: KS
authors.lastname: Sauter
authors.affiliation: Institute of Veterinary Virology, University of Bern, Bern, Switzerland.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mathys
authors.affiliation: Institute of Veterinary Virology, University of Bern, Bern, Switzerland.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Wyss
authors.affiliation: Institute of Veterinary Virology, University of Bern, Bern, Switzerland matthias.schweizer@vetsuisse.unibe.ch.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schweizer
mesh_terms: D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D001908:Diarrhea Viruses, Bovine Viral / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D006041:Goats; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007370:Interferon Type I / Q000037:antagonists & inhibitors*; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012756:Sheep; D013583:Synovial Membrane / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
chemical_list: D002966:Clathrin; D007370:Interferon Type I; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D004722:Endoribonucleases; C022537:ribonuclease E
references.citation: J Cell Biol. 1991 May;113(4):731-41
references.pmid: 1673969
references.citation: Virus Res. 2009 Mar;140(1-2):15-23
references.pmid: 19041350
references.citation: J Cell Biol. 1993 Dec;123(5):1107-17
references.pmid: 8245121
references.citation: Am J Physiol Lung Cell Mol Physiol. 2006 May;290(5):L946-55
references.pmid: 16361359
references.citation: J Clin Invest. 1973 Nov;52(11):2745-56
references.pmid: 4355998
references.citation: J Control Release. 2006 Nov 28;116(2):247-54
references.pmid: 16971016
references.citation: J Vet Med B Infect Dis Vet Public Health. 2004 Dec;51(10):427-32
references.pmid: 15606865
references.citation: Virology. 2007 Mar 1;359(1):6-18
references.pmid: 17046044
references.citation: Virology. 2005 Sep 30;340(2):265-76
references.pmid: 16043207
references.citation: J Gen Virol. 2003 May;84(Pt 5):1269-1274
references.pmid: 12692293
references.citation: In Vitro Cell Dev Biol Anim. 2010 May;46(5):403-7
references.pmid: 19997866
references.citation: Cell Logist. 2012 Oct 1;2(4):203-208
references.pmid: 23538558
references.citation: Mol Biol Cell. 1999 Apr;10(4):961-74
references.pmid: 10198050
references.citation: J Biol Chem. 2007 Nov 9;282(45):32730-41
references.pmid: 17848558
references.citation: J Virol. 1993 Jun;67(6):3288-94
references.pmid: 8388499
references.citation: Can J Comp Med. 1984 Apr;48(2):156-61
references.pmid: 6326980
references.citation: Virology. 2004 Dec 5;330(1):332-41
references.pmid: 15527858
references.citation: Mol Ther. 2010 Mar;18(3):561-9
references.pmid: 20010917
references.citation: Vet Rec. 1984 Jun 2;114(22):535-6
references.pmid: 6087539
references.citation: J Gen Virol. 1997 Nov;78 ( Pt 11):2779-87
references.pmid: 9367363
references.citation: J Virol. 2006 Jan;80(2):900-11
references.pmid: 16378992
references.citation: Biologicals. 2013 Jan;41(1):39-51
references.pmid: 22871358
references.citation: Structure. 2012 May 9;20(5):862-73
references.pmid: 22579253
references.citation: J Gen Virol. 2000 Feb;81(Pt 2):451-9
references.pmid: 10644844
references.citation: Annu Rev Anim Biosci. 2014 Feb;2:141-63
references.pmid: 25384138
references.citation: PLoS Pathog. 2013;9(6):e1003412
references.pmid: 23785283
references.citation: J Virol. 2004 Jan;78(1):136-45
references.pmid: 14671095
references.citation: Methods Enzymol. 2001;342:431-40
references.pmid: 11586914
references.citation: Science. 1993 Aug 27;261(5125):1169-71
references.pmid: 8356450
references.citation: Acta Pharmacol Toxicol (Copenh). 1974 Jan;34(1):27-32
references.pmid: 4406557
references.citation: J Virol. 2006 Apr;80(8):3912-22
references.pmid: 16571808
references.citation: J Gen Virol. 2008 Oct;89(Pt 10):2501-2506
references.pmid: 18796719
references.citation: J Virol. 2004 Feb;78(4):1792-9
references.pmid: 14747544
references.citation: Nat Immunol. 2004 Oct;5(10):987-95
references.pmid: 15454922
references.citation: J Biol Chem. 2002 Feb 15;277(7):5308-14
references.pmid: 11673454
references.citation: Vet Res. 2013 May 15;44:32
references.pmid: 23675947
references.citation: J Virol. 2006 Dec;80(23):11723-32
references.pmid: 16971436
references.citation: Methods Enzymol. 1984;108:336-47
references.pmid: 6084797
references.citation: J Virol. 2007 Apr;81(7):3327-38
references.pmid: 17215285
references.citation: J Virol. 2005 Apr;79(7):4191-200
references.pmid: 15767420
references.citation: Nature. 1982 Apr 15;296(5858):651-3
references.pmid: 7070509
references.citation: J Gen Virol. 2002 Sep;83(Pt 9):2153-2159
references.pmid: 12185268
references.citation: J Gen Virol. 2008 Sep;89(Pt 9):2147-2156
references.pmid: 18753224
references.citation: Immunity. 2011 Jul 22;35(1):135-45
references.pmid: 21723157
references.citation: Cell. 2006 Feb 24;124(4):783-801
references.pmid: 16497588
references.citation: J Virol. 2004 May;78(10):5507-12
references.pmid: 15113930
references.citation: J Virol. 1996 Jan;70(1):352-8
references.pmid: 8523547
references.citation: J Virol Methods. 2006 Oct;137(1):51-7
references.pmid: 16854474
references.citation: Proc Natl Acad Sci U S A. 1980 May;77(5):2706-10
references.pmid: 6156455
references.citation: J Virol. 2005 Aug;79(16):10826-9
references.pmid: 16051874
references.citation: J Cell Biol. 2001 Aug 6;154(3):535-47
references.pmid: 11481344
references.citation: J Virol. 2005 Sep;79(18):11901-13
references.pmid: 16140766
references.citation: PLoS Pathog. 2010 Mar 26;6(3):e1000829
references.pmid: 20360967
references.citation: Semin Immunol. 2007 Feb;19(1):33-40
references.pmid: 17336545
references.citation: Eur J Biochem. 1989 Dec 8;186(1-2):17-22
references.pmid: 2598927
references.citation: Microbiol Immunol. 1995;39(12):979-85
references.pmid: 8789057