title: NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies.
abstract: GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis.
  
  
  OBJECTIVE
  Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.
authors.affiliation: Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France CNRS UMR3569, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France.
authors.forename: Célia
authors.initials: C
authors.lastname: Boukadida
authors.affiliation: Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France CNRS UMR3569, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Marnata
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Lyon, France CNRS UMR 5086, Lyon, France Labex Ecofect, University of Lyon, Lyon, France.
authors.forename: Roland
authors.initials: R
authors.lastname: Montserret
authors.affiliation: Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France CNRS UMR3569, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France.
authors.forename: Lisette
authors.initials: L
authors.lastname: Cohen
authors.affiliation: Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France CNRS UMR3569, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Blumen
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gouttenoire
authors.affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.affiliation: Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Lyon, France CNRS UMR 5086, Lyon, France Labex Ecofect, University of Lyon, Lyon, France.
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.affiliation: Institut Pasteur, Unit of Molecular Genetics of RNA Viruses, Paris, France CNRS UMR3569, Paris, France Université Paris Diderot-Sorbonne Paris Cité, Paris, France annette.martin@pasteur.fr.
authors.forename: Annette
authors.initials: A
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D018178:Flaviviridae Infections / Q000378:metabolism* / Q000821:virology; D005455:Fluorescent Antibody Technique; D029406:GB virus B / Q000502:physiology; D016174:Hepacivirus / Q000502:physiology; D006526:Hepatitis C / Q000378:metabolism* / Q000821:virology; D006525:Hepatitis, Viral, Human / Q000378:metabolism* / Q000821:virology; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C097010:NS2 protein, Hepatitis C virus; D017361:Viral Nonstructural Proteins
references.citation: J Virol. 1995 Jul;69(7):4127-36
references.pmid: 7769671
references.citation: Nature. 2006 Aug 17;442(7104):831-5
references.pmid: 16862121
references.citation: Nat Med. 2005 Jul;11(7):791-6
references.pmid: 15951748
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5342-7
references.pmid: 19282477
references.citation: PLoS One. 2012;7(8):e42481
references.pmid: 22870331
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9962-7
references.pmid: 12907703
references.citation: PLoS One. 2009;4(2):e4419
references.pmid: 19204793
references.citation: PLoS Pathog. 2013;9(6):e1003438
references.pmid: 23818848
references.citation: J Viral Hepat. 2012 Mar;19(3):189-98
references.pmid: 22329373
references.citation: Electrophoresis. 1997 Dec;18(15):2714-23
references.pmid: 9504803
references.citation: PLoS Pathog. 2009 May;5(5):e1000403
references.pmid: 19412343
references.citation: Emerg Infect Dis. 2012 Dec;18(12):1976-82
references.pmid: 23171728
references.citation: Virology. 2003 Aug 1;312(2):270-80
references.pmid: 12919733
references.citation: Proc Natl Acad Sci U S A. 2013 May 14;110(20):8194-9
references.pmid: 23610427
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D363-6
references.pmid: 17142229
references.citation: J Biol Chem. 2002 Feb 8;277(6):4261-70
references.pmid: 11706032
references.citation: PLoS Pathog. 2007 Jul;3(7):e103
references.pmid: 17658949
references.citation: J Mol Biol. 2000 Sep 8;302(1):205-17
references.pmid: 10964570
references.citation: Science. 1999 Jul 2;285(5424):110-3
references.pmid: 10390360
references.citation: J Biol Chem. 2006 Sep 29;281(39):29221-7
references.pmid: 16882659
references.citation: Hepatology. 2005 May;41(5):986-94
references.pmid: 15793797
references.citation: Protein Eng. 1997 Jun;10(6):673-6
references.pmid: 9278280
references.citation: J Virol. 2007 Dec;81(23):13168-79
references.pmid: 17881454
references.citation: J Virol. 2006 Feb;80(4):1734-41
references.pmid: 16439530
references.citation: mBio. 2013 Apr 09;4(2):e00216-13
references.pmid: 23572554
references.citation: J Virol. 2007 Aug;81(16):8374-83
references.pmid: 17537845
references.citation: J Mol Graph. 1996 Feb;14(1):51-5, 29-32
references.pmid: 8744573
references.citation: J Virol. 1995 Sep;69(9):5621-30
references.pmid: 7637008
references.citation: J Virol. 2009 Sep;83(17):8379-95
references.pmid: 19515772
references.citation: J Virol. 2002 Aug;76(15):7736-46
references.pmid: 12097587
references.citation: Histochem Cell Biol. 2008 Sep;130(3):561-6
references.pmid: 18512067
references.citation: PLoS Pathog. 2010 Dec 16;6(12):e1001233
references.pmid: 21187906
references.citation: Trends Biochem Sci. 2000 Mar;25(3):147-50
references.pmid: 10694887
references.citation: Biochemistry. 2013 Aug 6;52(31):5295-303
references.pmid: 23841474
references.citation: J Virol. 2002 Dec;76(24):13001-14
references.pmid: 12438626
references.citation: Front Microbiol. 2011 Dec 07;2:240
references.pmid: 22319510
references.citation: J Biomol NMR. 1995 Jan;5(1):14-24
references.pmid: 7881270
references.citation: J Biol Chem. 2004 Sep 24;279(39):40835-43
references.pmid: 15247283
references.citation: J Biol Chem. 2004 Jun 11;279(24):24965-75
references.pmid: 15060070
references.citation: Nat Cell Biol. 2007 Sep;9(9):1089-97
references.pmid: 17721513
references.citation: J Virol. 2009 Jun;83(12):6257-68
references.pmid: 19357161
references.citation: Nat Rev Microbiol. 2013 Jul;11(7):482-96
references.pmid: 23748342
references.citation: J Virol. 2013 Apr;87(8):4130-45
references.pmid: 23408609
references.citation: J Virol. 2012 Jun;86(11):6171-8
references.pmid: 22491452
references.citation: EMBO J. 2002 Aug 1;21(15):3980-8
references.pmid: 12145199
references.citation: Trends Microbiol. 2011 Feb;19(2):95-103
references.pmid: 21146993
references.citation: J Virol. 2013 Aug;87(16):8971-81
references.pmid: 23740998
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11608-13
references.pmid: 21610165
references.citation: Virology. 1999 Sep 1;261(2):216-26
references.pmid: 10497107
references.citation: Virology. 2003 Jun 20;311(1):72-80
references.pmid: 12832204
references.citation: J Virol. 2014 Jun;88(11):6519-23
references.pmid: 24648458
references.citation: Nat Struct Biol. 1996 Oct;3(10):842-8
references.pmid: 8836100
references.citation: J Virol. 2002 Mar;76(6):2997-3006
references.pmid: 11861865
references.citation: J Virol. 2010 Nov;84(21):11580-4
references.pmid: 20739529
references.citation: J Virol. 2004 Feb;78(4):2062-71
references.pmid: 14747571
references.citation: J Gen Virol. 2010 Mar;91(Pt 3):727-33
references.pmid: 19906942
references.citation: J Virol. 1993 Aug;67(8):4665-75
references.pmid: 8392606
references.citation: J Virol. 1997 Jul;71(7):4985-9
references.pmid: 9188562
references.citation: J Med Virol. 2001 Dec;65(4):694-7
references.pmid: 11745933
references.citation: J Biol Chem. 2008 Oct 17;283(42):28546-62
references.pmid: 18644781
references.citation: PLoS Pathog. 2011 Jul;7(7):e1002144
references.pmid: 21814513
references.citation: J Virol. 2011 Feb;85(4):1706-17
references.pmid: 21147927
references.citation: J Magn Reson. 2003 Jan;160(1):65-73
references.pmid: 12565051
references.citation: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W668-73
references.pmid: 15215473
references.citation: J Virol. 2011 Jan;85(1):86-97
references.pmid: 20962101
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14545-50
references.pmid: 18799730
references.citation: J Virol. 2007 Jan;81(2):964-76
references.pmid: 17093192
references.citation: Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3401-5
references.pmid: 7724574
references.citation: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references.pmid: 7984417
references.citation: Nature. 2005 Oct 20;437(7062):1167-72
references.pmid: 16177806
references.citation: PLoS Pathog. 2011 Feb 10;7(2):e1001278
references.pmid: 21347350
references.citation: J Mol Biol. 2002 Mar 15;317(1):131-44
references.pmid: 11916384
references.citation: Protein Sci. 2009 Dec;18(12):2624-8
references.pmid: 19785006
references.citation: Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10583-7
references.pmid: 8248148