title: X-ray structure of the pestivirus NS3 helicase and its conformation in solution.
abstract: Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication.
  
  
  OBJECTIVE
  Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior--a characteristic probably shared with NS3 helicases from all Flaviviridae members--that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.
authors: Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France CNRS, UMR 3569, Paris, France tortoric@pasteur.fr rey@pasteur.fr.
authors: M Alejandra
authors: MA
authors: Tortorici
authors: Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France CNRS, UMR 3569, Paris, France Université Paris Sud, Gif sur Yvette, France.
authors: Stéphane
authors: S
authors: Duquerroy
authors: Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France Department of Physiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong.
authors: Jane
authors: J
authors: Kwok
authors: Global Phasing Ltd., Cambridge, United Kingdom.
authors: Clemens
authors: C
authors: Vonrhein
authors: Synchrotron Soleil, Gif sur Yvette, France.
authors: Javier
authors: J
authors: Perez
authors: Institute of Virology, University of Veterinary Medicine, Vienna, Austria.
authors: Benjamin
authors: B
authors: Lamp
authors: Global Phasing Ltd., Cambridge, United Kingdom.
authors: Gerard
authors: G
authors: Bricogne
authors: Institute of Virology, University of Veterinary Medicine, Vienna, Austria.
authors: Till
authors: T
authors: Rümenapf
authors: Institut de Biologie Intégrative de la Cellule, UMR 9198, Gif-sur-Yvette, France.
authors: Patrice
authors: P
authors: Vachette
authors: Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France CNRS, UMR 3569, Paris, France tortoric@pasteur.fr rey@pasteur.fr.
authors: Félix A
authors: FA
authors: Rey
mesh_terms: D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009841:Oligonucleotides / Q000235:genetics; D010576:Pestivirus / Q000201:enzymology*; D011487:Protein Conformation; D020365:RNA Helicases / Q000737:chemistry; D053838:Scattering, Small Angle; D012697:Serine Endopeptidases / Q000737:chemistry; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D009841:Oligonucleotides; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
references: PLoS One. 2011;6(9):e24257
references: 21977225
references: FEBS Lett. 2009 Feb 18;583(4):691-6
references: 19166847
references: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references: 20383002
references: Nat Rev Drug Discov. 2005 Oct;4(10):845-53
references: 16184083
references: Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):521-8
references: 20080715
references: J Mol Biol. 2007 Sep 14;372(2):444-55
references: 17658551
references: J Biol Chem. 2003 Nov 7;278(45):44514-24
references: 12944414
references: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W610-4
references: 15215461
references: Structure. 2012 May 9;20(5):862-73
references: 22579253
references: Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):72-82
references: 16369096
references: J Virol. 2012 Jun;86(11):6171-8
references: 22491452
references: Cell Rep. 2013 Jan 31;3(1):30-5
references: 23273918
references: J Virol. 1995 Mar;69(3):1720-6
references: 7853509
references: Nature. 2011 Nov 17;479(7373):423-7
references: 21947008
references: Nat Struct Biol. 1997 Jun;4(6):463-7
references: 9187654
references: Acta Crystallogr D Biol Crystallogr. 2006 Sep;62(Pt 9):1002-11
references: 16929101
references: J Virol. 2008 Aug;82(15):7624-39
references: 18508894
references: J Synchrotron Radiat. 2006 Mar;13(Pt 2):120-30
references: 16495612
references: J Biol Chem. 1998 Jun 12;273(24):15045-52
references: 9614113
references: Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W52-6
references: 16845062
references: EMBO J. 2008 Dec 3;27(23):3209-19
references: 19008861
references: Annu Rev Biochem. 2007;76:23-50
references: 17506634
references: PLoS Pathog. 2013;9(10):e1003704
references: 24146623
references: Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):6805-10
references: 23569276
references: Nat Struct Mol Biol. 2007 Jul;14(7):647-52
references: 17558417
references: J Biol Chem. 2010 Jun 11;285(24):18817-27
references: 20375022
references: BMC Struct Biol. 2011;11:6
references: 21269479
references: Nucleic Acids Res. 2014 Jul;42(Web Server issue):W320-4
references: 24753421
references: J Virol. 2008 Jan;82(1):173-83
references: 17942558
references: J Virol. 2011 Apr;85(7):3607-20
references: 21270154
references: PLoS Pathog. 2012;8(3):e1002598
references: 22457622
references: Biochem Biophys Res Commun. 2012 Jan 6;417(1):84-7
references: 22138238
references: Toxins (Basel). 2012 Aug;4(8):580-604
references: 23012649
references: Biochem Biophys Res Commun. 1998 Mar 6;244(1):35-40
references: 9514871
references: Structure. 2013 Jun 4;21(6):929-38
references: 23643950
references: Virology. 2008 Apr 10;373(2):426-36
references: 18201743
references: J Biol Chem. 2008 Oct 31;283(44):29929-37
references: 18723512
references: J Synchrotron Radiat. 2009 May;16(Pt 3):368-75
references: 19395800
references: Nucleic Acids Res. 2004;32(13):4060-70
references: 15289579
references: Proc Natl Acad Sci U S A. 2004 Mar 30;101(13):4425-30
references: 15070734
references: J Virol. 2013 Nov;87(21):11872-83
references: 23986594
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2210-21
references: 15572774
references: J Virol. 2008 Apr;82(7):3342-52
references: 18199634
references: J Virol. 2002 May;76(10):4773-84
references: 11967294
references: J Virol. 2007 Jan;81(2):629-38
references: 17079282
references: Virus Res. 2009 Apr;141(1):63-70
references: 19185595
references: Structure. 1999 Nov 15;7(11):1353-63
references: 10574797
references: J Virol. 2009 Dec;83(24):12895-906
references: 19793813
references: J Virol. 2013 Jun;87(12):7185-90
references: 23596285
references: J Virol. 2005 Aug;79(16):10278-88
references: 16051821
references: PeerJ. 2013 Jun 11;1:e85
references: 23781407
references: J Mol Biol. 2011 Feb 4;405(5):1139-53
references: 21145896
references: J Biol Chem. 2010 Jul 23;285(30):22725-31
references: 20457607
references: J Biol Chem. 2007 Nov 30;282(48):34913-20
references: 17921146
references: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references: 20124692
references: Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17345-50
references: 20855599
references: Arch Virol. 2007;152(8):1565-73
references: 17447110
references: J Virol. 2011 Dec;85(23):12351-61
references: 21957313
references: J Mol Biol. 1993 Dec 20;234(4):946-50
references: 8263940