Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture


HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.


Vyšlo v časopise: Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture. PLoS Pathog 6(5): e32767. doi:10.1371/journal.ppat.1000910
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000910

Souhrn

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.


Zdroje

1. SimmondsP

BukhJ

CombetC

DeleageG

EnomotoN

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

2. LindenbachBD

EvansMJ

SyderAJ

WolkB

TellinghuisenTL

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

3. WakitaT

PietschmannT

KatoT

DateT

MiyamotoM

2005 Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11 91 96

4. HellerT

SaitoS

AuerbachJ

WilliamsT

MoreenTR

2005 An in vitro model of hepatitis C virion production. Proc Natl Acad Sci USA 102 2579 2583

5. LindenbachBD

PlossA

VanwolleghemT

SyderAJ

McKeatingJA

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

6. ZhongJ

GastaminzaP

ChengG

KapadiaS

KatoT

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

7. SumpterRJr

LooY

FoyE

LiK

YoneyamaM

2005 Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 79 2689 2699

8. GuoJ

YanR

XuG

LiW

ZhengC

2009 Construction of the Vero cell culture system that can produce infectious HCV particles. Mol Biol Rep 36 111 120

9. 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

10. YiM

VillanuevaRA

ThomasDL

WakitaT

LemonSM

2006 Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 103 2310 2315

11. GottweinJM

ScheelTK

HoeghAM

LademannJB

Eugen-OlsenJ

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

12. JensenTB

GottweinJM

ScheelTK

HoeghAM

Eugen-OlsenJ

2008 Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis 198 1756 1765

13. ScheelTK

GottweinJM

JensenTB

PrentoeJC

HoeghAM

2008 Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci U S A 105 997 1002

14. GottweinJM

ScheelTK

JensenTB

LademannJB

PrentoeJC

2009 Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49 364 377

15. MateuG

DonisRO

WakitaT

BukhJ

GrakouiA

2008 Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death. Virology 376 397 407

16. PietschmannT

ZayasM

MeulemanP

LongG

AppelN

2009 Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog 5 e1000475

17. AlyHH

WatashiK

HijikataM

KanekoH

TakadaY

2007 Serum-derived hepatitis C virus infectivity in interferon regulatory factor-7-suppressed human primary hepatocytes. J Hepatol 46 26 36

18. FarquharMJ

McKeatingJA

2008 Primary hepatocytes as targets for hepatitis C virus replication. J Viral Hepat 15 849 854

19. JacobsBL

LanglandJO

1996 When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219 339 349

20. TaylorDR

PuigM

DarnellME

MihalikK

FeinstoneSM

2005 New antiviral pathway that mediates hepatitis C virus replicon interferon sensitivity through ADAR1. J Virol 79 6291 6298

21. MorseDP

BassBL

1997 Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36 8429 8434

22. O'MalleyRP

MarianoTM

SiekierkaJ

MathewsMB

1986 A mechanism for the control of protein synthesis by adenovirus VA RNAI. Cell 44 391 400

23. LeiM

LiuY

SamuelCE

1998 Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase. Virology 245 188 196

24. DiazMO

ZieminS

Le BeauMM

PithaP

SmithSD

1988 Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines. Proc Natl Acad Sci U S A 85 5259 5263

25. MoscaJD

PithaPM

1986 Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Mol Cell Biol 6 2279 2283

26. ClarkePA

Pe'eryT

MaY

MathewsMB

1994 Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI. Nucleic Acids Res 22 4364 4374

27. GunneryS

MathewsMB

1995 Functional mRNA can be generated by RNA polymerase III. Mol Cell Biol 15 3597 3607

28. SaldanhaJ

HeathA

LelieN

PisaniG

NublingM

2000 Calibration of HCV working reagents for NAT assays against the HCV international standard. The Collaborative Study Group. Vox Sang 78 217 224

29. YuMW

FarshidM

MasonBL

TanD

KochWH

1998 Formulation of a hepatitis C virus RNA panel for standardization of nucleic acid testing of blood, plasma, and their derived products. Hepatology 28 566A

30. KolykhalovAA

AgapovEV

BlightKJ

MihalikK

FeinstoneSM

1997 Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277 570 574

31. ReedLJ

MuenchH

1938 A simple method of estimating fifty percent end points. Am J Hyg 27 493 497

32. CormierEG

TsamisF

KajumoF

DursoRJ

GardnerJP

2004 CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 101 7270 7274

33. FlintM

Loomis-PriceLD

ShottonC

DubuissonJ

MonkP

1999 Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol 73 6235 6244

34. PileriP

UematsuY

CampagnoliS

GalliG

FalugiF

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

35. YuMY

BartoschB

ZhangP

GuoZP

RenziPM

2004 Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma. Proc Natl Acad Sci U S A 101 7705 7710

36. 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

37. Le PogamS

JiangWR

LevequeV

RajyaguruS

MaH

2006 In vitro selected Con1 subgenomic replicons resistant to 2′-C-methyl-cytidine or to R1479 show lack of cross resistance. Virology 351 349 359

38. ChevalierC

SaulnierA

BenureauY

FlechetD

DelgrangeD

2007 Inhibition of hepatitis C virus infection in cell culture by small interfering RNAs. Mol Ther 15 1452 1462

39. GermiR

CranceJM

GarinD

GuimetJ

Lortat-JacobH

2002 Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption. J Med Virol 68 206 215

40. DiamondDL

SyderAJ

JacobsJM

SorensenCM

WaltersKA

2010 Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLoS Pathog 6 e1000719

41. PetrikJ

ParkerH

AlexanderGJ

1999 Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA. J Gen Virol 80 (Pt 12) 3109 3113

42. RomanowskiT

SikorskaK

BielawskiKP

2008 GUS and PMM1 as suitable reference genes for gene expression analysis in the liver tissue of patients with chronic hepatitis. Med Sci Monit 14 BR147 BR152

43. WaxmanS

WurmbachE

2007 De-regulation of common housekeeping genes in hepatocellular carcinoma. BMC Genomics 8 243

44. von HahnT

RiceCM

2008 Hepatitis C virus entry. J Biol Chem 283 3689 3693

45. IroM

WitteveldtJ

AngusAG

WoerzI

KaulA

2009 A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication. Antiviral Res

46. WaltersKA

SyderAJ

LedererSL

DiamondDL

PaeperB

2009 Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes. PLoS Pathog 5 e1000269

47. SaitoT

OwenDM

JiangF

MarcotrigianoJ

GaleMJr

2008 Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature 454 523 527

48. SaundersLR

BarberGN

2003 The dsRNA binding protein family: critical roles, diverse cellular functions. FASEB J 17 961 983

49. TaylorDR

LeeSB

RomanoPR

MarshakDR

HinnebuschAG

1996 Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR. Mol Cell Biol 16 6295 6302

50. ClemensMJ

1997 PKR–a protein kinase regulated by double-stranded RNA. Int J Biochem Cell Biol 29 945 949

51. ScaddenAD

SmithCW

1997 A ribonuclease specific for inosine-containing RNA: a potential role in antiviral defence? EMBO J 16 2140 2149

52. ScaddenAD

SmithCW

2001 Specific cleavage of hyper-edited dsRNAs. EMBO J 20 4243 4252

53. EspertL

DegolsG

GongoraC

BlondelD

WilliamsBR

2003 ISG20, a new interferon-induced RNase specific for single-stranded RNA, defines an alternative antiviral pathway against RNA genomic viruses. J Biol Chem 278 16151 16158

54. TaylorDR

SilbersteinE

2009 Innate immunity and hepatitis C virus: eluding the host cell defense. Front Biosci 14 4950 4961

55. PawlotskyJM

2003 Mechanisms of antiviral treatment efficacy and failure in chronic hepatitis C. Antiviral Res 59 1 11

56. GaleMJJr

KorthMJ

TangNM

TanSL

HopkinsDA

1997 Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230 217 227

57. TaylorDR

ShiST

RomanoPR

BarberGN

LaiMM

1999 Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285 107 110

58. VyasJ

EliaA

ClemensMJ

2003 Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA. RNA 9 858 870

59. ZhangP

WuCG

MihalikK

Virata-TheimerML

YuMY

2007 Hepatitis C virus epitope-specific neutralizing antibodies in Igs prepared from human plasma. Proc Natl Acad Sci U S A 104 8449 8454

60. LaemmliUK

1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680 685

61. MajorME

MihalikK

FernandezJ

SeidmanJ

KleinerD

1999 Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus. J Virol 73 3317 3325

62. PuigM

MihalikK

YuMY

FeinstoneSM

MajorME

2002 Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay. J Virol Methods 105 253 263

63. OkamotoH

OkadaS

SugiyamaY

KuraiK

IizukaH

1991 Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. J Gen Virol 72 (Pt 11) 2697 2704

64. RandallG

GrakouiA

RiceCM

2003 Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci U S A 100 235 240

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