#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells


We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.


Vyšlo v časopise: RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells. PLoS Pathog 6(4): e32767. doi:10.1371/journal.ppat.1000885
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000885

Souhrn

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.


Zdroje

1. LemonS

WalkerC

AlterM

YiM

2007 Hepatitis C virus.

KnipeD

HowleyP

Fields Virology 5 ed Philadelphia, PA Lippincott-Raven Publishers 1253 1304

2. WasleyA

AlterMJ

2000 Epidemiology of hepatitis C: geographic differences and temporal trends. Semin Liver Dis 20 1 16

3. GrakouiA

WychowskiC

LinC

FeinstoneSM

RiceCM

1993 Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67 1385 1395

4. HijikataM

MizushimaH

TanjiY

KomodaY

HirowatariY

1993 Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90 10773 10777

5. Tsukiyama-KoharaK

IizukaN

KoharaM

NomotoA

1992 Internal ribosome entry site within hepatitis C virus RNA. J Virol 66 1476 1483

6. TanakaT

KatoN

ChoMJ

ShimotohnoK

1995 A novel sequence found at the 3′ terminus of hepatitis C virus genome. Biochem Biophys Res Commun 215 744 749

7. YouS

StumpDD

BranchAD

RiceCM

2004 A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J Virol 78 1352 1366

8. 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

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. LindenbachBD

EvansMJ

SyderAJ

WolkB

TellinghuisenTL

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

11. 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

12. MurayamaA

DateT

MorikawaK

AkazawaD

MiyamotoM

2007 The NS3 helicase and NS5B-to-3′X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. J Virol 81 8030 8040

13. WengL

DuJ

ZhouJ

DingJ

WakitaT

2009 Modification of hepatitis C virus 1b RNA polymerase to make a highly active JFH1-type polymerase by mutation of the thumb domain. Arch Virol 154 765 773

14. FriebeP

BartenschlagerR

2002 Genetic analysis of sequences in the 3′ nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 76 5326 5338

15. YanagiM

PurcellRH

EmersonSU

BukhJ

1999 Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology 262 250 263

16. YouS

RiceCM

2008 3′ RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 82 184 195

17. NakabayashiH

TaketaK

MiyanoK

YamaneT

SatoJ

1982 Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42 3858 3863

18. KuikenC

CombetC

BukhJ

ShinIT

DeleageG

2006 A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes. Hepatology 44 1355 1361

19. van den HoffMJ

MoormanAF

LamersWH

1992 Electroporation in ‘intracellular’ buffer increases cell survival. Nucleic Acids Res 20 2902

20. KatoT

DateT

MiyamotoM

SugiyamaM

TanakaY

2005 Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system. J Clin Microbiol 43 5679 5684

21. ZukerM

2003 Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31 3406 3415

22. FriebeP

BoudetJ

SimorreJP

BartenschlagerR

2005 Kissing-loop interaction in the 3′ end of the hepatitis C virus genome essential for RNA replication. J Virol 79 380 392

23. LevequeVJ

JohnsonRB

ParsonsS

RenJ

XieC

2003 Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis. J Virol 77 9020 9028

24. ZhongW

FerrariE

LesburgCA

MaagD

GhoshSK

2000 Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74 9134 9143

25. HongZ

CameronCE

WalkerMP

CastroC

YaoN

2001 A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase. Virology 285 6 11

26. SimisterP

SchmittM

GeitmannM

WichtO

DanielsonUH

2009 Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. J Virol

27. CaiZ

YiM

ZhangC

LuoG

2005 Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase. J Virol 79 11607 11617

28. ArumugaswamiV

RemenyiR

KanagavelV

SueEY

Ngoc HoT

2008 High-resolution functional profiling of hepatitis C virus genome. PLoS Pathog 4 e1000182 doi:10.1371/journal.ppat.1000182

29. KanaiA

TanabeK

KoharaM

1995 Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase. FEBS Lett 376 221 224

30. LuoG

HamatakeRK

MathisDM

RacelaJ

RigatKL

2000 De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J Virol 74 851 863

31. HuangL

HwangJ

SharmaSD

HargittaiMR

ChenY

2005 Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J Biol Chem 280 36417 36428

32. GontarekRR

GutshallLL

HeroldKM

TsaiJ

SatheGM

1999 hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3′NTR of the HCV RNA genome. Nucleic Acids Res 27 1457 1463

33. LuoG

1999 Cellular proteins bind to the poly(U) tract of the 3′ untranslated region of hepatitis C virus RNA genome. Virology 256 105 118

34. 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 (Pt12) 3109 3113

35. SpangbergK

WiklundL

SchwartzS

2001 Binding of the La autoantigen to the hepatitis C virus 3′ untranslated region protects the RNA from rapid degradation in vitro. J Gen Virol 82 113 120

36. SpangbergK

WiklundL

SchwartzS

2000 HuR, a protein implicated in oncogene and growth factor mRNA decay, binds to the 3′ ends of hepatitis C virus RNA of both polarities. Virology 274 378 390

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2010 Číslo 4
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

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#