A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

English version České info

Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.

Vyšlo v časopise: A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly. PLoS Pathog 7(7): e32767. doi:10.1371/journal.ppat.1002144
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002144

Souhrn

Zdroje

1. SimmondsPBukhJCombetCDeleageGEnomotoN 2005 Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42 962 973

2. EvansMJvon HahnTTscherneDMSyderAJPanisM 2007 Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446 801 805

3. PileriPUematsuYCampagnoliSGalliGFalugiF 1998 Binding of hepatitis C virus to CD81. Science 282 938 941

4. PlossAEvansMJGaysinskayaVAPanisMYouH 2009 Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457 882 886

5. ScarselliEAnsuiniHCerinoRRoccaseccaRMAcaliS 2002 The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21 5017 5025

6. GonzalezMECarrascoL 2003 Viroporins. FEBS Lett 552 28 34

7. JonesCTMurrayCLEastmanDKTasselloJRiceCM 2007 Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81 8374 8383

8. SteinmannEPeninFKallisSPatelAHBartenschlagerR 2007 Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions. PLoS Pathog 3 e103

9. WozniakALGriffinSRowlandsDHarrisMYiM 2010 Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production. PLoS Pathog 6 e1001087

10. SakaiAClaireMSFaulkKGovindarajanSEmersonSU 2003 The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences. Proc Natl Acad Sci U S A 100 11646 11651

11. LindenbachBDThielHJRiceCM 2007 Flaviviridae: the viruses and their replication. KnipeDMHowleyPM Fields Virology. 5th ed Philadelphia Lippincott-Raven 1101 1152

12. RoingeardPHouriouxCBlanchardEBrandDAit-GoughoulteM 2004 Hepatitis C virus ultrastructure and morphogenesis. Biol Cell 96 103 108

13. GastaminzaPChengGWielandSZhongJLiaoW 2008 Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 82 2120 2129

14. DeleersnyderVPillezAWychowskiCBlightKXuJ 1997 Formation of native hepatitis C virus glycoprotein complexes. J Virol 71 697 704

15. RouilleYHelleFDelgrangeDRoingeardPVoissetC 2006 Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J Virol 80 2832 2841

16. BarbaGHarperFHaradaTKoharaMGoulinetS 1997 Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc Natl Acad Sci U S A 94 1200 1205

17. BoulantSTargett-AdamsPMcLauchlanJ 2007 Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus. J Gen Virol 88 2204 2213

18. BoulantSDouglasMWMoodyLBudkowskaATargett-AdamsP 2008 Hepatitis C virus core protein induces lipid droplet redistribution in a microtubule- and dynein-dependent manner. Traffic 9 1268 1282

19. MiyanariYAtsuzawaKUsudaNWatashiKHishikiT 2007 The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9 1089 1097

20. ShavinskayaABoulantSPeninFMcLauchlanJBartenschlagerR 2007 The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J Biol Chem 282 37158 37169

21. MurphySMartinSPartonRG 2009 Lipid droplet-organelle interactions; sharing the fats. Biochim Biophys Acta 1791 441 447

22. OzekiSChengJTauchi-SatoKHatanoNTaniguchiH 2005 Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane. J Cell Sci 118 2601 2611

23. Targett-AdamsPHopeGBoulantSMcLauchlanJ 2008 Maturation of hepatitis C virus core protein by signal peptide peptidase is required for virus production. J Biol Chem 283 16850 16859

24. BoulantSMontserretRHopeRGRatinierMTargett-AdamsP 2006 Structural determinants that target the hepatitis C virus core protein to lipid droplets. J Biol Chem 281 22236 22247

25. RoingeardPHouriouxCBlanchardEPrensierG 2008 Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy. Histochem Cell Biol 130 561 566

26. WakitaTPietschmannTKatoTDateTMiyamotoM 2005 Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11 791 796

27. LindenbachBDEvansMJSyderAJWolkBTellinghuisenTL 2005 Complete replication of hepatitis C virus in cell culture. Science 309 623 626

28. ZhongJGastaminzaPChengGKapadiaSKatoT 2005 Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102 9294 9299

29. LindenbachBDMeulemanPPlossAVanwolleghemTSyderAJ 2006 Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci U S A 103 3805 3809

30. PietschmannTKaulAKoutsoudakisGShavinskayaAKallisS 2006 Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci U S A 103 7408 7413

31. MajeauNFromentinRSavardCDuvalMTremblayMJ 2009 Palmitoylation of hepatitis C virus core protein is important for virion production. J Biol Chem 284 33915 33925

32. MoradpourDEnglertCWakitaTWandsJR 1996 Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222 51 63

33. NakamuraNAkashiTTanedaTKogoHKikuchiA 2004 ADRP is dissociated from lipid droplets by ARF1-dependent mechanism. Biochem Biophys Res Commun 322 957 965

34. WolinsNEQuaynorBKSkinnerJRSchoenfishMJTzekovA 2005 S3-12, Adipophilin, and TIP47 package lipid in adipocytes. J Biol Chem 280 19146 19155

35. BrasaemleDLDoliosGShapiroLWangR 2004 Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J Biol Chem 279 46835 46842

36. BartzRZehmerJKZhuMChenYSerreroG 2007 Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. J Proteome Res 6 3256 3265

37. MartinSPartonRG 2006 Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7 373 378

38. RobenekHHofnagelOBuersIRobenekMJTroyerD 2006 Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis. J Cell Sci 119 4215 4224

39. KaulAWoerzIMeulemanPLeroux-RoelsGBartenschlagerR 2007 Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 81 13168 13179

40. GottweinJMScheelTKHoeghAMLademannJBEugen-OlsenJ 2007 Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 133 1614 1626

41. BartenschlagerRPietschmannT 2005 Efficient hepatitis C virus cell culture system: what a difference the host cell makes. Proc Natl Acad Sci U S A 102 9739 9740

42. JiraskoVMontserretRAppelNJanvierAEustachiL 2008 Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 283 28546 28562

43. StaplefordKALindenbachBD 2011 Hepatitis C virus NS2 coordinates virus particle assembly through physical interactions with the E1–E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 85 1706 1717

44. SandrinVCossetFL 2006 Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein. J Biol Chem 281 528 542

45. JiraskoVMontserretRLeeJYGouttenoireJMoradpourD 2010 Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly. PLoS Pathog 6 e1001233

46. MurrayCLJonesCTTasselloJRiceCM 2007 Alanine scanning of the hepatitis C virus core protein reveals numerous residues essential for production of infectious virus. J Virol 81 10220 10231

47. McLauchlanJ 2009 Hepatitis C virus: viral proteins on the move. Biochem Soc Trans 37 986 990

48. GriffinSDBealesLPClarkeDSWorsfoldOEvansSD 2003 The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535 34 38

49. PavlovicDNevilleDCArgaudOBlumbergBDwekRA 2003 The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100 6104 6108

50. PremkumarAWilsonLEwartGDGagePW 2004 Cation-selective ion channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 557 99 103

51. GriffinSDHarveyRClarkeDSBarclayWSHarrisM 2004 A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 85 451 461

52. MontserretRSaintNVanbelleCSalvayAGSimorreJP 2010 NMR structure and ion channel activity of the p7 protein from hepatitis C virus. J Biol Chem 285 31446 31461

53. ClarkeDGriffinSBealesLGelaisCSBurgessS 2006 Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro. J Biol Chem 281 37057 37068

54. LuikPChewCAittoniemiJChangJWentworthPJr 2009 The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy. Proc Natl Acad Sci U S A 106 12712 12716

55. PatargiasGZitzmannNDwekRFischerWB 2006 Protein-protein interactions: modeling the hepatitis C virus ion channel p7. J Med Chem 49 648 655

56. Carrere-KremerSMontpellier-PalaCCocquerelLWychowskiCPeninF 2002 Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76 3720 3730

57. GriffinSClarkeDMcCormickCRowlandsDHarrisM 2005 Signal peptide cleavage and internal targeting signals direct the hepatitis C virus p7 protein to distinct intracellular membranes. J Virol 79 15525 15536

58. HaqshenasGMackenzieJMDongXGowansEJ 2007 Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome. J Gen Virol 88 134 142

59. PopescuCICallensNTrinelDRoingeardPMoradpourD 2011 NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly. PLoS Pathog 7 e1001278

60. BrohmCSteinmannEFrieslandMLorenzICPatelA 2009 Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation. J Virol 83 11682 11693

61. HaradaTTautzNThielHJ 2000 E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J Virol 74 9498 9506

62. YamagaAKOuJH 2002 Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem 277 33228 33234

63. DentzerTGLorenzICEvansMJRiceCM 2009 Determinants of the hepatitis C virus nonstructural protein 2 protease domain required for production of infectious virus. J Virol 83 12702 12713

64. YiMMaYYatesJLemonSM 2009 Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation. PLoS Pathog 5 e1000403

65. YiMMaYYatesJLemonSM 2007 Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol 81 629 638

66. PhanTBeranRKPetersCLorenzICLindenbachBD 2009 Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes. J Virol 83 8379 8395

67. MaYAnantpadmaMTimpeJMShanmugamSSinghSM 2011 Hepatitis C Virus NS2 Protein Serves as a Scaffold for Virus Assembly by Interacting with both Structural and Nonstructural Proteins. J Virol 85 86 97

68. TedburyPWelbournSPauseAKingBGriffinS 2011 The subcellular localization of the hepatitis C virus non-structural protein NS2 is regulated by an ion channel-independent function of the p7 protein. J Gen Virol 92 819 830

69. LavilletteDTarrAWVoissetCDonotPBartoschB 2005 Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus. Hepatology 41 265 274

70. DreuxMDao ThiVLFresquetJGuerinMJuliaZ 2009 Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains. PLoS Pathog 5 e1000310

71. KoutsoudakisGHerrmannEKallisSBartenschlagerRPietschmannT 2007 The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells. J Virol 81 588 598

72. GastaminzaPKapadiaSBChisariFV 2006 Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles. J Virol 80 11074 11081

73. SandrinVBosonBSalmonPGayWNegreD 2002 Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates. Blood 100 823 832

74. NegreDMangeotPEDuisitGBlanchardSVidalainPO 2000 Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther 7 1613 1623

75. OhsakiYShinoharaYSuzukiMFujimotoT 2010 A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy. Histochem Cell Biol 133 477 480

76. OhsakiYMaedaTFujimotoT 2005 Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins. Histochem Cell Biol 124 445 452

77. FukumotoSFujimotoT 2002 Deformation of lipid droplets in fixed samples. Histochem Cell Biol 118 423 428

78. DunnKWKamockaMMMcDonaldJH 2011 A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol 300 C723 C742

79. KolesnikovaLBambergSBerghoferBBeckerS 2004 The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway. J Virol 78 2382 2393