#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly


Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.


Vyšlo v časopise: NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly. PLoS Pathog 7(2): e32767. doi:10.1371/journal.ppat.1001278
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1001278

Souhrn

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.


Zdroje

1. WasleyA

AlterMJ

2000

Epidemiology of hepatitis C: geographic differences and temporal trends.

Semin Liver Dis

20

1

16

2. FeldJJ

HoofnagleJH

2005

Mechanism of action of interferon and ribavirin in treatment of hepatitis C.

Nature

436

967

972

3. LindenbachBD

ThielHJ

RiceCM

2007

Flaviviridae: the viruses and their replication.

KnipeDM

HowleyPM

Fields Virology. 5th ed

Philadelphia, Pa

Lippincott Williams & Wilkins

1101

1152

4. MoradpourD

PeninF

RiceCM

2007

Replication of hepatitis C virus.

Nat Rev Microbiol

5

453

463

5. BlightKJ

McKeatingJA

RiceCM

2002

Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.

J Virol

76

13001

13014

6. LohmannV

KörnerF

KochJ-O

HerianU

TheilmannL

1999

Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.

Science

285

110

113

7. WakitaT

PietschmannT

KatoT

DateT

MiyamotoM

2005

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.

Nat Med

11

791

796

8. LindenbachBD

EvansMJ

SyderAJ

WolkB

TellinghuisenTL

2005

Complete replication of hepatitis C virus in cell culture.

Science

309

623

626

9. ZhongJ

GastaminzaP

ChengG

KapadiaS

KatoT

2005

Robust hepatitis C virus infection in vitro.

Proc Natl Acad Sci U S A

102

9294

9299

10. McLauchlanJ

2009

Lipid droplets and hepatitis C virus infection.

Biochim Biophys Acta

1791

552

559

11. RouilléY

HelleF

DelgrangeD

RoingeardP

VoissetC

2006

Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus.

J Virol

80

2832

2841

12. MiyanariY

AtsuzawaK

UsudaN

WatashiK

HishikiT

2007

The lipid droplet is an important organelle for hepatitis C virus production.

Nat Cell Biol

9

1089

1097

13. BoulantS

Targett-AdamsP

McLauchlanJ

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

14. AppelN

ZayasM

MillerS

Krijnse-LockerJ

SchallerT

2008

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly.

PLoS Pathog

4

e1000035

15. EvansMJ

RiceCM

GoffSP

2004

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.

Proc Natl Acad Sci U S A

101

13038

13043

16. MasakiT

SuzukiR

MurakamiK

AizakiH

IshiiK

2008

Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles.

J Virol

82

7964

7976

17. TellinghuisenTL

FossKL

TreadawayJ

2008

Regulation of hepatitis C virion production via phosphorylation of the NS5A protein.

PLoS Pathog

4

e1000032

18. DentzerTG

LorenzIC

EvansMJ

RiceCM

2009

Determinants of the hepatitis C virus nonstructural protein 2 protease domain required for production of infectious virus.

J Virol

83

12702

12713

19. JiraskoV

MontserretR

AppelN

JanvierA

EustachiL

2008

Structural and functional characterization of non-structural protein 2 for its role in hepatitis C virus assembly.

J Biol Chem

283

28546

28562

20. JonesCT

MurrayCL

EastmanDK

TasselloJ

RiceCM

2007

Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.

J Virol

81

8374

8383

21. PietschmannT

KaulA

KoutsoudakisG

ShavinskayaA

KallisS

2006

Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.

Proc Natl Acad Sci U S A

103

7408

7413

22. YiM

MaY

YatesJ

LemonSM

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

23. PhanT

BeranRK

PetersC

LorenzIC

LindenbachBD

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

24. Carrère-KremerS

Montpellier-PalaC

CocquerelL

WychowskiC

PeninF

2002

Subcellular localization and topology of the p7 polypeptide of hepatitis C virus.

J Virol

76

3720

3730

25. CocquerelL

Op de BeeckA

LambotM

RousselJ

DelgrangeD

2002

Topologic changes in the transmembrane domains of hepatitis C virus envelope glycoproteins.

Embo J

21

2893

2902

26. BraunP

PerssonB

KabackR

von HeijneG

1997

Alanine insertion scanning mutagenesis of lactose permease transmembrane helices.

J Biol Chem

272

29566

29571

27. MingarroI

WhitleyP

LemmonM

von HeijneG

1996

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix : A rapid way to map helix-helix ineractions in integral membrane protein.

Protein Sci

5

1339

1341

28. Op De BeeckA

MolenkampR

CaronM

Ben YounesA

BredenbeekP

2003

Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope.

J Virol

77

813

820

29. Op De BeeckA

MontserretR

DuvetS

CocquerelL

CacanR

2000

Role of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 in the assembly of the noncovalent E1E2 heterodimer.

J Biol Chem

275

31428

31437

30. DelgrangeD

PillezA

CastelainS

CocquerelL

RouilléY

2007

Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins.

J Gen Virol

88

2495

2503

31. GoueslainL

AlsalehK

HorellouP

RoingeardP

DescampsV

2010

Identification of GBF1 as a cellular factor required for hepatitis C virus RNA replication.

J Virol

84

773

787

32. SteinmannE

PeninF

KallisS

PatelAH

BartenschlagerR

2007

Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.

PLoS Pathog

3

e103

33. SelbyMJ

GlazerE

MasiarzF

HoughtonM

1994

Complex processing and protein∶protein interactions in the E2∶NS2 region of HCV.

Virology

204

114

122

34. YiM

MaY

YatesJ

LemonSM

2009

Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation.

PLoS Pathog

5

e1000403

35. GriffinSD

HarveyR

ClarkeDS

BarclayWS

HarrisM

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

36. SchweizerA

FransenJAM

BächiT

GinselL

HauriH-P

1988

Identification, by a monoclonal antibody, of a 53-kD protein associated with tubulo-vesicular compartment at the cis-side of the Golgi apparatus.

J Cell Biol

107

1643

1653

37. ShimoiW

EzawaI

NakamotoK

UesakiS

GabreskiG

2005

p125 is localized in endoplasmic reticulum exit sites and involved in their organization.

J Biol Chem

280

10141

10148

38. ShugrueCA

KolenER

PetersH

CzernikA

KaiserC

1999

Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat.

J Cell Sci

112

4547

4556

39. MaY

AnantpadmaM

TimpeJM

ShanmugamS

SinghSM

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

40. MurrayCL

JonesCT

RiceCM

2008

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis.

Nat Rev Microbiol

6

699

708

41. BoulantS

MontserretR

HopeG

RatinierM

Targett-AdamsP

2006

Structural determinants that target the hepatitis C virus core protein to lipid droplets.

J Biol Chem

281

22236

22247

42. VieyresG

ThomasX

DescampsV

DuverlieG

PatelAH

2010

Characterization of the envelope glycoproteins associated with infectious hepatitis C virus.

J Virol

84

10159

10168

43. VillanuevaR

RouilléY

DubuissonJ

2005

Interactions between virus proteins and host cell membranes.

Int Rev Cytol

245

171

244

44. HaqshenasG

MackenzieJM

DongX

GowansEJ

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

45. SprietC

TrinelD

WaharteF

DesleeD

VandenbunderB

2007

Correlated fluorescence lifetime and spectral measurements in living cells.

Microsc Res Tech

70

85

94

46. YamadaH

Padilla-ParraS

ParkSJ

ItohT

ChaineauM

2009

Dynamic interaction of amphiphysin with N-WASP regulates actin assembly.

J Biol Chem

284

34244

34256

47. ZhangW

ChipmanPR

CorverJ

JohnsonPR

ZhangY

2003

Visualization of membrane protein domains by cryo-electron microscopy of dengue virus.

Nat Struct Biol

10

907

912

48. JonesDM

PatelAH

Targett-AdamsP

McLauchlanJ

2009

The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus.

J Virol

83

2163

2177

49. PavlovicD

NevilleDC

ArgaudO

BlumbergB

DwekRA

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. LiljestromP

LusaS

HuylebroeckD

GaroffH

1991

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.

J Virol

65

4107

4113

51. LoewyA

SmythJ

von BonsdorffCH

LiljestromP

SchlesingerMJ

1995

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

J Virol

69

469

475

52. CorseE

MachamerCE

2003

The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction.

Virology

312

25

34

53. FischerF

StegenCF

MastersPS

SamsonoffWA

1998

Analysis of constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly.

J Virol

72

7885

7894

54. VennemaH

GodekeGJ

RossenJW

VoorhoutWF

HorzinekMC

1996

Nucleocapsid-independent assembly of coronavirus-like particles by co- expression of viral envelope protein genes.

Embo J

15

2020

2028

55. NakabayashiH

TaketaK

MiyanoK

YamaneT

SatoJ

1982

Growth of human hepatoma cells lines with differentiated functions in chemically defined medium.

Cancer Res

42

3858

3863

56. DubuissonJ

HsuHH

CheungRC

GreenbergHB

RussellDG

1994

Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.

J Virol

68

6147

6160

57. FlintM

MaidensC

Loomis-PriceLD

ShottonC

DubuissonJ

1999

Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.

J Virol

73

6235

6244

58. MaillardP

KrawczynskiK

NitkiewiczJ

BronnertC

SidorkiewiczM

2001

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

J Virol

75

8240

8250

59. Rocha-PeruginiV

MontpellierC

DelgrangeD

WychowskiC

HelleF

2008

The CD81 partner EWI-2wint inhibits hepatitis C virus entry.

PLoS ONE

3

e1866

60. O'ConnorDV

Phillips

1984

Time-correlated single photon counting

London

Academic Press

61. WaharteF

SprietC

HeliotL

2006

Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

Cytometry A

69

299

306

62. DraperNR

SmithH

1998

Applied regression analysis

New York

Wiley-Interscience

63. ColemanTF

LiY

1994

On the convergence of reflective Newton methods for large-scale nonlinear minimization subject to bounds.

Mathematical Programming

67

189

224

64. LakowiczJR

1999

Principles of fluorescence Spectroscopy

New York

Kluwer Academic / Plenum Publishers

65. MederackeI

WedemeyerH

CiesekS

SteinmannE

RaupachR

2009

Performance and clinical utility of a novel fully automated quantitative HCV-core antigen assay.

J Clin Virol

46

210

215

66. MorotaK

FujinamiR

KinukawaH

MachidaT

OhnoK

2009

A new sensitive and automated chemiluminescent microparticle immunoassay for quantitative determination of hepatitis C virus core antigen.

J Virol Methods

157

8

14

67. CastelainS

DescampsV

ThibaultV

FrancoisC

BonteD

2004

TaqMan amplification system with an internal positive control for HCV RNA quantitation.

J Clin Virol

31

227

234

68. ShapiroSS

WilkMB

1965

An analysis of variance test for normality.

Biometrika

52

591

611

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2011 Číslo 2
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#