Hepatitis C Virus RNA Replication Depends on Specific and -Acting Activities of Viral Nonstructural Proteins

English version České info

RNA viruses encode one or more proteins that form an RNA replicase, which serves to propagate the viral RNA genome. Some replication genes can function in trans, meaning that proteins expressed by one viral genome can complement and assist in the replication of a defective mutant virus within the same cell. Other viral genes work in cis, meaning that they are unable to complement defective mutants; these cis-acting genes therefore link the replication of a viral RNA genome to its translation. Because genetic complementation reflects the formation of molecular interactions that were previously absent or nonfunctional, understanding the cis- and trans-activities of viral replication proteins can provide insights into the structure and function of viral replicases. Here we develop a novel methodology to study interactions between components of the viral replicase to define essential cis- and trans-activities for all five hepatitis C virus (HCV) replication gene products. Our studies show that representative defects in each HCV replication protein can be complemented in trans. However, specific features of NS3 and NS5B were required in cis, indicating that these proteins specifically function to replicate the genome that encodes them. These data define important activities of the HCV replication proteins and provide new insight into how these proteins function together as an enzyme complex.

Vyšlo v časopise: Hepatitis C Virus RNA Replication Depends on Specific and -Acting Activities of Viral Nonstructural Proteins. PLoS Pathog 11(4): e32767. doi:10.1371/journal.ppat.1004817
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004817

Souhrn

Zdroje

1. Eigen M, Schuster P (1979) The hypercycle. A principle of natural self-organization. Heidelberg: Springer-Verlag. 92 p.

2. Crowder S, Kirkegaard K (2005) Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat Genet 37: 701–709. 15965477

3. Li JP, Baltimore D (1988) Isolation of poliovirus 2C mutants defective in viral RNA synthesis. J Virol 62: 4016–4021. 2845120

4. Tanner EJ, Liu HM, Oberste MS, Pallansch M, Collett MS, et al. (2014) Dominant drug targets suppress the emergence of antiviral resistance. Elife 3.

5. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R (2006) Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439: 344–348. 16327776

6. Huang AS, Baltimore D (1970) Defective viral particles and viral disease processes. Nature 226: 325–327. 5439728

7. van der Most RG, Luytjes W, Rutjes S, Spaan WJ (1995) Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus. J Virol 69: 3744–3751. 7745722

8. Molenkamp R, Greve S, Spaan WJ, Snijder EJ (2000) Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs. J Virol 74: 9062–9070. 10982351

9. Su YP, Fan YH, Brian DA (2014) Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis. J Virol 88: 8868–8882. doi: 10.1128/JVI.00738-14 24872586

10. Yi G, Kao C (2008) cis- and trans-acting functions of brome mosaic virus protein 1a in genomic RNA1 replication. J Virol 82: 3045–3053. 18160434

11. Novak JE, Kirkegaard K (1994) Coupling between genome translation and replication in an RNA virus. Genes Dev 8: 1726–1737. 7958852

12. Collis PS, O'Donnell BJ, Barton DJ, Rogers JA, Flanegan JB (1992) Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells. J Virol 66: 6480–6488. 1328676

13. Grassmann CW, Isken O, Tautz N, Behrens SE (2001) Genetic analysis of the pestivirus nonstructural coding region: defects in the NS5A unit can be complemented in trans. J Virol 75: 7791–7802. 11483722

14. Khromykh AA, Sedlak PL, Westaway EG (2000) cis- and trans-acting elements in flavivirus RNA replication. J Virol 74: 3253–3263. 10708442

15. Paul AV, Rieder E, Kim DW, van Boom JH, Wimmer E (2000) Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J Virol 74: 10359–10370. 11044080

16. Goodfellow I, Chaudhry Y, Richardson A, Meredith J, Almond JW, et al. (2000) Identification of a cis-acting replication element within the poliovirus coding region. J Virol 74: 4590–4600. 10775595

17. Friebe P, Boudet J, Simorre JP, Bartenschlager R (2005) Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential for RNA replication. J Virol 79: 380–392. 15596831

18. Lee H, Shin H, Wimmer E, Paul AV (2004) cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome. J Virol 78: 10865–10877. 15452207

19. You S, Stump DD, Branch AD, Rice CM (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. 14722290

20. Liao CL, Lai MM (1995) A cis-acting viral protein is not required for the replication of a coronavirus defective-interfering RNA. Virology 209: 428–436. 7778278

21. Lin J, Guo J, Finer J, Dorrance AE, Redinbaugh MG, et al. (2014) The bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation. J Virol 88: 3213–3222. doi: 10.1128/JVI.03301-13 24390330

22. Lindenbach BD, Murray CL, Thiel HJ, Rice CM (2013) Flaviviridae. In: Knipe DM, Howley PM, editors. Fields Virology. Sixth ed. Philadelphia: Lippincott Williams & Wilkins. pp. 712–746.

23. Bartenschlager R, Lohmann V, Penin F (2013) The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 11: 482–496. doi: 10.1038/nrmicro3046 23748342

24. Lindenbach BD, Rice CM (2013) The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol 11: 688–700. doi: 10.1038/nrmicro3098 24018384

25. Lohmann V, Korner F, Koch JO, Herian U, Theilmann L, et al. (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285: 110–113. 10390360

26. Bartenschlager R, Frese M, Pietschmann T (2004) Novel insights into hepatitis C virus replication and persistence. Adv Virus Res 63: 71–180. 15530561

27. Ranjith-Kumar CT, Kao CC (2006) Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase. In: Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK).

28. Steinkühler C (2004) Hepacivirin. In: Barrett AJ, Rawlings ND, Woessner JF, editors. Handbook of proteolytic enzymes. 2nd ed. London: Elsevier. pp. 1773–1779.

29. Li XD, Sun L, Seth RB, Pineda G, Chen ZJ (2005) Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A 102: 17717–17722. 16301520

30. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, et al. (2005) Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437: 1167–1172. 16177806

31. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon AC, et al. (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A 102: 2992–2997. 15710891

32. Kang X, Chen X, He Y, Guo D, Guo L, et al. (2013) DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication. Virology 435: 385–394. doi: 10.1016/j.virol.2012.10.025 23137809

33. Morikawa K, Gouttenoire J, Hernandez C, Dao Thi VL, Tran HT, et al. (2014) Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease. Hepatology 59: 423–433. doi: 10.1002/hep.26671 23929719

34. Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM (2000) Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol 74: 2046–2051. 10644379

35. Lam AM, Frick DN (2006) Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase. J Virol 80: 404–411. 16352565

36. Pyle AM (2008) Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys 37: 317–336. doi: 10.1146/annurev.biophys.37.032807.125908 18573084

37. Beran RK, Lindenbach BD, Pyle AM (2009) The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase. J Virol 83: 3268–3275. doi: 10.1128/JVI.01849-08 19153239

38. Tanji Y, Hijikata M, Satoh S, Kaneko T, Shimotohno K (1995) Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing. J Virol 69: 1575–1581. 7853491

39. Gouttenoire J, Penin F, Moradpour D (2010) Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev Med Virol 20: 117–129. doi: 10.1002/rmv.640 20069613

40. Paul D, Romero-Brey I, Gouttenoire J, Stoitsova S, Krijnse-Locker J, et al. (2011) NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes. J Virol 85: 6963–6976. doi: 10.1128/JVI.00502-11 21543474

41. Ross-Thriepland D, Harris M (2014) Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on! J Gen Virol.

42. Verdegem D, Badillo A, Wieruszeski JM, Landrieu I, Leroy A, et al. (2011) Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A. J Biol Chem 286: 20441–20454. doi: 10.1074/jbc.M110.182436 21489988

43. Grise H, Frausto S, Logan T, Tang H (2012) A conserved tandem cyclophilin-binding site in hepatitis C virus nonstructural protein 5A regulates Alisporivir susceptibility. J Virol 86: 4811–4822. doi: 10.1128/JVI.06641-11 22345441

44. Hanoulle X, Badillo A, Wieruszeski JM, Verdegem D, Landrieu I, et al. (2009) Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J Biol Chem 284: 13589–13601. doi: 10.1074/jbc.M809244200 19297321

45. Fernandes F, Ansari IU, Striker R (2010) Cyclosporine inhibits a direct interaction between cyclophilins and hepatitis C NS5A. PLoS One 5: e9815. doi: 10.1371/journal.pone.0009815 20352119

46. Foster TL, Gallay P, Stonehouse NJ, Harris M (2011) Cyclophilin A Interacts with Domain II of Hepatitis C Virus NS5A and Stimulates RNA Binding in an Isomerase-Dependent Manner. J Virol 85: 7460–7464. doi: 10.1128/JVI.00393-11 21593166

47. Reiss S, Rebhan I, Backes P, Romero-Brey I, Erfle H, et al. (2011) Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe 9: 32–45. doi: 10.1016/j.chom.2010.12.002 21238945

48. Evans MJ, Rice CM, Goff SP (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. 15326295

49. Hamamoto I, Nishimura Y, Okamoto T, Aizaki H, Liu M, et al. (2005) Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J Virol 79: 13473–13482. 16227268

50. Tu H, Gao L, Shi ST, Taylor DR, Yang T, et al. (1999) Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263: 30–41. 10544080

51. Brass V, Gouttenoire J, Wahl A, Pal Z, Blum HE, et al. (2010) Hepatitis C Virus RNA Replication Requires a Conserved Structural Motif within the Transmembrane Domain of the NS5B RNA-Dependent RNA Polymerase. J Virol 84: 11580–11584. doi: 10.1128/JVI.01519-10 20739529

52. Moradpour D, Brass V, Bieck E, Friebe P, Gosert R, et al. (2004) Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication. J Virol 78: 13278–13284. 15542678

53. Adair R, Patel AH, Corless L, Griffin S, Rowlands DJ, et al. (2009) Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA. J Gen Virol 90: 833–842. doi: 10.1099/vir.2008.006049-0 19223490

54. Ishii K, Murakami K, Hmwe SS, Zhang B, Li J, et al. (2008) Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins. Biochem Biophys Res Commun 371: 446–450. doi: 10.1016/j.bbrc.2008.04.110 18445476

55. Pacini L, Graziani R, Bartholomew L, De Francesco R, Paonessa G (2009) Naturally occurring hepatitis C virus subgenomic deletion mutants replicate efficiently in Huh-7 cells and are trans-packaged in vitro to generate infectious defective particles. J Virol 83: 9079–9093. doi: 10.1128/JVI.00308-09 19587042

56. Steinmann E, Brohm C, Kallis S, Bartenschlager R, Pietschmann T (2008) Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J Virol 82: 7034–7046. doi: 10.1128/JVI.00118-08 18480457

57. Appel N, Herian U, Bartenschlager R (2005) Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79: 896–909. 15613318

58. Jones DM, Patel AH, Targett-Adams P, McLauchlan J (2009) The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83: 2163–2177. doi: 10.1128/JVI.01885-08 19073716

59. Fridell RA, Qiu D, Valera L, Wang C, Rose RE, et al. (2011) Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052. J Virol 85: 7312–7320. doi: 10.1128/JVI.00253-11 21593143

60. Fridell RA, Valera L, Qiu D, Kirk MJ, Wang C, et al. (2013) Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles. J Virol 87: 2320–2329. doi: 10.1128/JVI.02861-12 23236071

61. Tong X, Malcolm BA (2006) Trans-complementation of HCV replication by non-structural protein 5A. Virus Res 115: 122–130. 16146661

62. Herod MR, Schregel V, Hinds C, Liu M, McLauchlan J, et al. (2014) Genetic complementation of hepatitis C virus nonstructural protein functions associated with replication exhibits requirements that differ from those for virion assembly. J Virol 88: 2748–2762. doi: 10.1128/JVI.03588-13 24352463

63. Krieger N, Lohmann V, Bartenschlager R (2001) Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75: 4614–4624. 11312331

64. Lohmann V (2009) HCV replicons: overview and basic protocols. Methods Mol Biol 510: 145–163. doi: 10.1007/978-1-59745-394-3_11 19009259

65. Phan T, Kohlway A, Dimberu P, Pyle AM, Lindenbach BD (2011) The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly. J Virol 85: 1193–1204. doi: 10.1128/JVI.01889-10 21047963

66. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, et al. (2003) Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125: 1808–1817. 14724833

67. Lesburg CA, Cable MB, Ferrari E, Hong Z, Mannarino AF, et al. (1999) Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 6: 937–943. 10504728

68. Kim YC, Russell WK, Ranjith-Kumar CT, Thomson M, Russell DH, et al. (2005) Functional analysis of RNA binding by the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 280: 38011–38019. 16166071

69. Mosley RT, Edwards TE, Murakami E, Lam AM, Grice RL, et al. (2012) Structure of hepatitis C virus polymerase in complex with primer-template RNA. J Virol 86: 6503–6511. doi: 10.1128/JVI.00386-12 22496223

70. Grakoui A, McCourt DW, Wychowski C, Feinstone SM, Rice CM (1993) Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol 67: 2832–2843. 8386278

71. Tomei L, Failla C, Santolini E, De Francesco R, La Monica N (1993) NS3 is a serine protease required for processing of hepatitis C virus polyprotein. Journal of virology 67: 4017–4026. 7685406

72. Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA, et al. (1998) Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 6: 89–100. 9493270

73. Lin C, Kim JL (1999) Structure-based mutagenesis study of hepatitis C virus NS3 helicase. J Virol 73: 8798–8807. 10482634

74. Appleby TC, Anderson R, Fedorova O, Pyle AM, Wang R, et al. (2011) Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV. J Mol Biol 405: 1139–1153. doi: 10.1016/j.jmb.2010.11.034 21145896

75. Preugschat F, Danger DP, Carter LH 3rd, Davis RG, Porter DJ (2000) Kinetic analysis of the effects of mutagenesis of W501 and V432 of the hepatitis C virus NS3 helicase domain on ATPase and strand-separating activity. Biochemistry 39: 5174–5183. 10819985

76. Gu M, Rice CM (2010) Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc Natl Acad Sci U S A 107: 521–528. doi: 10.1073/pnas.0913380107 20080715

77. Kim JW, Seo MY, Shelat A, Kim CS, Kwon TW, et al. (2003) Structurally conserved amino Acid W501 is required for RNA helicase activity but is not essential for DNA helicase activity of hepatitis C virus NS3 protein. J Virol 77: 571–582. 12477861

78. Kohlway A, Pirakitikulr N, Ding SC, Yang F, Luo D, et al. (2014) The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus. J Virol 88: 10970–10974. doi: 10.1128/JVI.00745-14 24965468

79. Lindenbach BD, Prágai BM, Montserret R, Beran RK, Pyle AM, et al. (2007) The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J Virol 81: 8905–8918. 17581983

80. Kohlway A, Pirakitikulr N, Barrera FN, Potapova O, Engelman DM, et al. (2014) Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain. Journal of Virology 88: 628–642. doi: 10.1128/JVI.02052-13 24173222

81. Appel N, Pietschmann T, Bartenschlager R (2005) Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol 79: 3187–3194. 15709040

82. Blight KJ, Kolykhalov AA, Rice CM (2000) Efficient initiation of HCV RNA replication in cell culture. Science 290: 1972–1974. 11110665

83. Evans MJ, Rice CM, Goff SP (2004) Genetic interactions between hepatitis C virus replicons. J Virol 78: 12085–12089. 15479852

84. Petrakova O, Volkova E, Gorchakov R, Paessler S, Kinney RM, et al. (2005) Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells. J Virol 79: 7597–7608. 15919912

85. Agapov EV, Frolov I, Lindenbach BD, Prägai BM, Schlesinger S, et al. (1998) Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proc Natl Acad Sci USA 95: 12989–12994. 9789028

86. Khromykh AA, Sedlak PL, Guyatt KJ, Hall RA, Westaway EG (1999) Efficient trans-complementation of the flavivirus kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase. J Virol 73: 10272–10280. 10559344

87. Lindenbach BD, Rice CM (1997) trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71: 9608–9617. 9371625

88. Lindenbach BD, Rice CM (1999) Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol 73: 4611–4621. 10233920

89. Yi M, Ma Y, Yates J, Lemon SM (2009) Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation. PLoS Pathog 5: e1000403. doi: 10.1371/journal.ppat.1000403 19412343

90. Gao M (2013) Antiviral activity and resistance of HCV NS5A replication complex inhibitors. Curr Opin Virol.

91. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, et al. (2010) Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465: 96–100. doi: 10.1038/nature08960 20410884

92. Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M, et al. (2014) Daclatasvir-Like Inhibitors of NS5A Block Early Biogenesis of Hepatitis C Virus-Induced Membranous Replication Factories, Independent of RNA Replication. Gastroenterology 147: 1094–1105 e1025. doi: 10.1053/j.gastro.2014.07.019 25046163

93. National Center for Biotechnology Information PubChem BioAssay Database; CID = 25154714, Source = Bristol Meyers Squibb Co.

94. Bachmair A, Finley D, Varshavasky A (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234: 179–186. 3018930

95. Baker RT, Tobias JW, Varshavsky A (1992) Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family. J Biol Chem 267: 23364–23375. 1429680

96. Phan T, Beran RK, Peters C, Lorenz IC, Lindenbach BD (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. doi: 10.1128/JVI.00891-09 19515772

97. Pietschmann T, Lohmann V, Kaul A, Krieger N, Rinck G, et al. (2002) Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76: 4008–4021. 11907240

98. Chen J, Noueiry A, Ahlquist P (2003) An alternate pathway for recruiting template RNA to the brome mosaic virus RNA replication complex. J Virol 77: 2568–2577. 12551995

99. Derbyshire KM, Grindley ND (1996) Cis preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation. Mol Microbiol 21: 1261–1272. 8898394

100. Duval-Valentin G, Chandler M (2011) Cotranslational control of DNA transposition: a window of opportunity. Mol Cell 44: 989–996. doi: 10.1016/j.molcel.2011.09.027 22195971

101. Burt DW, Brammar WJ (1982) The cis-specificity of the Q-gene product of bacteriophage lambda. Mol Gen Genet 185: 468–472. 6212756

102. Francke B, Ray DS (1972) Cis-limited action of the gene-A product of bacteriophage phiX174 and the essential bacterial site (E. coli-electron microscopy-cis-acting protein-specifically-nicked RF). Proc Natl Acad Sci U S A 69: 475–479. 4551145

103. Kulpa DA, Moran JV (2006) Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat Struct Mol Biol 13: 655–660. 16783376

104. Wei W, Gilbert N, Ooi SL, Lawler JF, Ostertag EM, et al. (2001) Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol 21: 1429–1439. 11158327

105. Spagnolo JF, Rossignol E, Bullitt E, Kirkegaard K (2010) Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays. RNA 16: 382–393. doi: 10.1261/rna.1955410 20051491

106. Pata JD, Schultz SC, Kirkegaard K (1995) Functional oligomerization of poliovirus RNA-dependent RNA polymerase. Rna 1: 466–477. 7489508

107. Hobson SD, Rosenblum ES, Richards OC, Richmond K, Kirkegaard K, et al. (2001) Oligomeric structures of poliovirus polymerase are important for function. EMBO J 20: 1153–1163. 11230138

108. Wang QM, Hockman MA, Staschke K, Johnson RB, Case KA, et al. (2002) Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase. J Virol 76: 3865–3872. 11907226

109. Asabe SI, Tanji Y, Satoh S, Kaneko T, Kimura K, et al. (1997) The N-terminal region of hepatitis C virus-encoded NS5A is important for NS4A-dependent phosphorylation. J Virol 71: 790–796. 8985418

110. Kaneko T, Tanji Y, Satoh S, Hijikata M, Asabe S, et al. (1994) Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun 205: 320–326. 7999043

111. Koch JO, Bartenschlager R (1999) Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 73: 7138–7146. 10438800

112. Neddermann P, Clementi A, De Francesco R (1999) Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol 73: 9984–9991. 10559312

113. Bernardin F, Stramer SL, Rehermann B, Page-Shafer K, Cooper S, et al. (2007) High levels of subgenomic HCV plasma RNA in immunosilent infections. Virology 365: 446–456. 17493654

114. Noppornpanth S, Smits SL, Lien TX, Poovorawan Y, Osterhaus AD, et al. (2007) Characterization of hepatitis C virus deletion mutants circulating in chronically infected patients. J Virol 81: 12496–12503. 17728237

115. Sugiyama K, Suzuki K, Nakazawa T, Funami K, Hishiki T, et al. (2009) Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro. J Virol 83: 6922–6928. doi: 10.1128/JVI.02674-08 19369330

116. Delang L, Neyts J, Vliegen I, Abrignani S, Neddermann P, et al. (2013) Hepatitis C virus-specific directly acting antiviral drugs. Curr Top Microbiol Immunol 369: 289–320. doi: 10.1007/978-3-642-27340-7_12 23463206

117. Sulkowski MS, Gardiner DF, Rodriguez-Torres M, Reddy KR, Hassanein T, et al. (2014) Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. N Engl J Med 370: 211–221. doi: 10.1056/NEJMoa1306218 24428467

118. Grassmann CW, Isken O, Behrens SE (1999) Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study. J Virol 73: 9196–9205. 10516027

119. Tai CL, Pan WC, Liaw SH, Yang UC, Hwang LH, et al. (2001) Structure-based mutational analysis of the hepatitis C virus NS3 helicase. J Virol 75: 8289–8297. 11483774

120. Wang X, Lee WM, Watanabe T, Schwartz M, Janda M, et al. (2005) Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates. J Virol 79: 13747–13758. 16227294

121. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H (1993) Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 67: 3835–3844. 8389908

122. Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC (1999) Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure 7: 1353–1363. 10574797

123. Dumas E, Masante C, Astier-Gin T, Lapaillerie D, Ventura M (2007) The hepatitis C virus minigenome: a new cellular model for studying viral replication. J Virol Methods 142: 59–66. 17320981

124. King RW, Zecher M, Jeffries MW, Carroll DR, Parisi JM, et al. (2002) A cell-based model of HCV-negative-strand RNA replication utilizing a chimeric hepatitis C virus/reporter RNA template. Antivir Chem Chemother 13: 353–362. 12718407

125. Yang J, Lei YF, An QX, Yin W, Lu X, et al. (2010) Properties of hepatitis C virus minigenome containing mutated 5'UTR region and luciferase transgene. Acta Virol 54: 105–112. 20545439

126. Zhang J, Yamada O, Sakamoto T, Yoshida H, Araki H, et al. (2005) Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression. J Virol 79: 5923–5932. 15857978

127. Zhang J, Yamada O, Yoshida H, Sakamoto T, Araki H, et al. (2007) Helper virus-independent trans-replication of hepatitis C virus-derived minigenome. Biochem Biophys Res Commun 352: 170–176. 17112469

128. DeMarini DJ, Johnston VK, Konduri M, Gutshall LL, Sarisky RT (2003) Intracellular hepatitis C virus RNA-dependent RNA polymerase activity. J Virol Methods 113: 65–68. 14500128

129. Ranjith-Kumar CT, Wen Y, Baxter N, Bhardwaj K, Cheng Kao C (2011) A cell-based assay for RNA synthesis by the HCV polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by NS5A. PLoS One 6: e22575. doi: 10.1371/journal.pone.0022575 21799903

130. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL, et al. (2005) Complete replication of hepatitis C virus in cell culture. Science 309: 623–626. 15947137

131. Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83: 8122–8126. 3095828

132. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675. 22930834

133. Beran RK, Pyle AM (2008) Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem 283: 29929–29937. doi: 10.1074/jbc.M804065200 18723512

134. Harlow E, Lane DP (1988) Antibodies: a laboratory manual. In: Harlow E, Lane DP, editors. Antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.

135. Lindberg V (2000) Uncertainties and Error Propagation: Part I of a manual on Uncertainties, Graphing, and the Vernier Caliper.