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 LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 2
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Genetic Mapping Identifies Novel Highly Protective Antigens for an Apicomplexan Parasite
- Type I Interferon Signaling Regulates Ly6C Monocytes and Neutrophils during Acute Viral Pneumonia in Mice
- Infections in Cells: Transcriptomic Characterization of a Novel Host-Symbiont Interaction
- The ESCRT-0 Component HRS is Required for HIV-1 Vpu-Mediated BST-2/Tetherin Down-Regulation