1887

Journal of General Virology header logo

Volume 99, Issue 2

Self-association and conformational variation of NS5A domain 1 of hepatitis C virus Free

Abstract

Direct-acting antivirals (DAAs) targeting the non-structural 5A (NS5A) protein of the hepatitis C virus (HCV) are crucial drugs that have shown exceptional clinical success in patients. However, their mode of action (MoA) remains unclear, and drug-resistant HCV strains are rapidly emerging. It is critical to characterize the behaviour of the NS5A protein in solution, which can facilitate the development of new classes of inhibitors or improve the efficacy of the currently available DAAs. Using biophysical methods, including dynamic light scattering, size exclusion chromatography and chemical cross-linking experiments, we showed that the NS5A domain 1 from genotypes 1b and 1a of the HCV intrinsically self-associated and existed as a heterogeneous mixture in solution. Interestingly, the NS5A domain 1 from genotypes 1b and 1a exhibited different dynamic equilibria of monomers to higher-order structures. Using small-angle X-ray scattering, we studied the structural dynamics of the various states of the NS5A domain 1 in solution. We also tested the effect of daclatasvir (DCV), the most prominent DAA, on self-association of the wild and DCV-resistant mutant (Y93H) NS5A domain 1 proteins, and demonstrated that DCV induced the formation of large and irreversible protein aggregates that eventually precipitated out. This study highlights the conformational variability of the NS5A domain 1 of HCV, which may be an intrinsic structural behaviour of the HCV NS5A domain 1 in solution.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): conformational variation , daclatasvir , direct-acting antivirals , drug resistance , hepatitis C virus and NS5A protein
© 2018 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001000
2018-02-01
2024-04-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/99/2/194.html?itemId=/content/journal/jgv/10.1099/jgv.0.001000&mimeType=html&fmt=ahah

References

  1. WHO Global Health Sector Strategy on Viral Hepatitis 2016–2021: Towards Ending Viral Hepatitis Geneva, Switzerland: 2016
     [Google Scholar]
  2. Hajarizadeh B, Grebely J, Dore GJ. Epidemiology and natural history of HCV infection. Nat Rev Gastroenterol Hepatol 2013; 10:553–562 [View Article][PubMed]
     [Google Scholar]
  3. Heim MH. 25 years of interferon-based treatment of chronic hepatitis C: an epoch coming to an end. Nat Rev Immunol 2013; 13:535–542 [View Article][PubMed]
     [Google Scholar]
  4. Bidell MR, McLaughlin M, Faragon J, Morse C, Patel N. Desirable characteristics of hepatitis C treatment regimens: a review of what we have and what we need. Infect Dis Ther 2016; 5:299–312 [View Article][PubMed]
     [Google Scholar]
  5. Bertino G, Ardiri A, Proiti M, Rigano G, Frazzetto E et al. Chronic hepatitis C: this and the new era of treatment. World J Hepatol 2016; 8:92–106 [View Article][PubMed]
     [Google Scholar]
  6. Afdhal NH, Serfaty L. Effect of registries and cohort studies on HCV treatment. Gastroenterology 2016; 151:387–390 [View Article][PubMed]
     [Google Scholar]
  7. O'Boyle II DR, Nower PT, Gao M, Fridell R, Wang C et al. Synergistic activity of combined NS5A inhibitors. Antimicrob Agents Chemother 2015; 60:1573–1583 [View Article][PubMed]
     [Google Scholar]
  8. Asselah T, Marcellin P. Interferon free therapy with direct acting antivirals for HCV. Liver Int 2013; 33:93–104 [View Article][PubMed]
     [Google Scholar]
  9. Kaneko T, Tanji Y, Satoh S, Hijikata M, Asabe S et al. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun 1994; 205:320–326 [View Article][PubMed]
     [Google Scholar]
  10. Ross-Thriepland D, Harris M. Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation. J Virol 2014; 88:1421–1432 [View Article][PubMed]
     [Google Scholar]
  11. Tellinghuisen TL, Marcotrigiano J, Gorbalenya AE, Rice CM. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J Biol Chem 2004; 279:48576–48587 [View Article][PubMed]
     [Google Scholar]
  12. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 1995; 96:224–230 [View Article][PubMed]
     [Google Scholar]
  13. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010; 465:96–100 [View Article][PubMed]
     [Google Scholar]
  14. Ross-Thriepland D, Harris M. Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!. J Gen Virol 2015; 96:727–738 [View Article][PubMed]
     [Google Scholar]
  15. Appel N, Pietschmann T, Bartenschlager R. 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 2005; 79:3187–3194 [View Article][PubMed]
     [Google Scholar]
  16. Moradpour D, Evans MJ, Gosert R, Yuan Z, Blum HE et al. Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 2004; 78:7400–7409 [View Article][PubMed]
     [Google Scholar]
  17. Chung YL, Sheu ML, Yen SH. Hepatitis C virus NS5A as a potential viral Bcl-2 homologue interacts with Bax and inhibits apoptosis in hepatocellular carcinoma. Int J Cancer 2003; 107:65–73 [View Article][PubMed]
     [Google Scholar]
  18. Appel N, Zayas M, Miller S, Krijnse-Locker J, Schaller T et al. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 2008; 4:e1000035 [View Article][PubMed]
     [Google Scholar]
  19. Macdonald A, Crowder K, Street A, McCormick C, Harris M. The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity. J Gen Virol 2004; 85:721–729 [View Article][PubMed]
     [Google Scholar]
  20. Tripathi LP, Kambara H, Chen YA, Nishimura Y, Moriishi K et al. Understanding the biological context of NS5A-host interactions in HCV infection: a network-based approach. J Proteome Res 2013; 12:2537–2551 [View Article][PubMed]
     [Google Scholar]
  21. Penin F, Brass V, Appel N, Ramboarina S, Montserret R et al. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J Biol Chem 2004; 279:40835–40843 [View Article][PubMed]
     [Google Scholar]
  22. Liang Y, Ye H, Kang CB, Yoon HS. Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded. Biochemistry 2007; 46:11550–11558 [View Article][PubMed]
     [Google Scholar]
  23. Hanoulle X, Verdegem D, Badillo A, Wieruszeski JM, Penin F et al. Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded. Biochem Biophys Res Commun 2009; 381:634–638 [View Article][PubMed]
     [Google Scholar]
  24. Tellinghuisen TL, Marcotrigiano J, Rice CM. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005; 435:374–379 [View Article][PubMed]
     [Google Scholar]
  25. Love RA, Brodsky O, Hickey MJ, Wells PA, Cronin CN. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J Virol 2009; 83:4395–4403 [View Article][PubMed]
     [Google Scholar]
  26. Lambert SM, Langley DR, Garnett JA, Angell R, Hedgethorne K et al. The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors. Protein Sci 2014; 23:723–734 [View Article][PubMed]
     [Google Scholar]
  27. Kohler JJ, Nettles JH, Amblard F, Hurwitz SJ, Bassit L et al. Approaches to hepatitis C treatment and cure using NS5A inhibitors. Infect Drug Resist 2014; 7:41–56 [View Article][PubMed]
     [Google Scholar]
  28. Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013; 11:482–496 [View Article][PubMed]
     [Google Scholar]
  29. Barakat KH, Anwar-Mohamed A, Tuszynski JA, Robins MJ, Tyrrell DL et al. A refined model of the HCV NS5A protein bound to daclatasvir explains drug-resistant mutations and activity against divergent genotypes. J Chem Inf Model 2015; 55:362–373 [View Article][PubMed]
     [Google Scholar]
  30. Lim PJ, Chatterji U, Cordek D, Sharma SD, Garcia-Rivera JA et al. Correlation between NS5A dimerization and hepatitis C virus replication. J Biol Chem 2012; 287:30861–30873 [View Article][PubMed]
     [Google Scholar]
  31. Hwang J, Huang L, Cordek DG, Vaughan R, Reynolds SL et al. Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins. J Virol 2010; 84:12480–12491 [View Article][PubMed]
     [Google Scholar]
  32. Karahan HE, Karakuş K, Birer Ö. Simultaneous DLS–SLS study of titanium and titanium/silicon oxide sol growth. J Sol-Gel Sci Technol 2015; 76:251–259 [View Article]
     [Google Scholar]
  33. Stevens FJ. Analysis of protein-protein interaction by simulation of small-zone size exclusion chromatography. Stochastic formulation of kinetic rate contributions to observed high-performance liquid chromatography elution characteristics. Biophys J 1989; 55:1155–1167 [View Article][PubMed]
     [Google Scholar]
  34. Durand D, Vivès C, Cannella D, Pérez J, Pebay-Peyroula E et al. NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers. J Struct Biol 2010; 169:45–53 [View Article][PubMed]
     [Google Scholar]
  35. Svergun D, Barberato C, Koch MHJ. CRYSOL-a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 1995; 28:768–773 [View Article]
     [Google Scholar]
  36. Franke D, Jeffries CM, Svergun DI. Correlation map, a goodness-of-fit test for one-dimensional X-ray scattering spectra. Nat Methods 2015; 12:419–422 [View Article][PubMed]
     [Google Scholar]
  37. Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI. PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr 2003; 36:1277–1282 [View Article]
     [Google Scholar]
  38. Franke D, Svergun DI. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Crystallogr 2009; 42:342–346 [View Article][PubMed]
     [Google Scholar]
  39. O'Boyle II DR, Sun JH, Nower PT, Lemm JA, Fridell RA et al. Characterizations of HCV NS5A replication complex inhibitors. Virology 2013; 444:343–354 [View Article][PubMed]
     [Google Scholar]
  40. Mankouri J, Milward A, Pryde KR, Warter L, Martin A et al. A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B. J Gen Virol 2008; 89:1911–1920 [View Article][PubMed]
     [Google Scholar]
  41. Fridell RA, Wang C, Sun JH, O'Boyle II DR, Nower P et al. Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations. Hepatology 2011; 54:1924–1935 [View Article][PubMed]
     [Google Scholar]
  42. Fridell RA, Qiu D, Wang C, Valera L, Gao M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother 2010; 54:3641–3650 [View Article][PubMed]
     [Google Scholar]
  43. Gao M. Antiviral activity and resistance of HCV NS5A replication complex inhibitors. Curr Opin Virol 2013; 3:514–520 [View Article][PubMed]
     [Google Scholar]
  44. Bagaglio S, Andolina A, Merli M, Uberti-Foppa C, Morsica G. Frequency of natural resistance within NS5a replication complex domain in hepatitis C genotypes 1a, 1b: possible implication of subtype-specific resistance selection in multiple direct acting antivirals drugs combination treatment. Viruses 2016; 8:91 [View Article][PubMed]
     [Google Scholar]
  45. Sun JH, O'Boyle II DR, Zhang Y, Wang C, Nower P et al. Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5A replication complex inhibitor, BMS-790052. Hepatology 2012; 55:1692–1699 [View Article][PubMed]
     [Google Scholar]
  46. Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M et al. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication. Gastroenterology 2014; 147:1094–1105 [View Article][PubMed]
     [Google Scholar]
  47. Sun JH, O'Boyle II DR, Fridell RA, Langley DR, Wang C et al. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A. Nature 2015; 527:245–248 [View Article][PubMed]
     [Google Scholar]
  48. Bhattacharya D, Ansari IH, Hamatake R, Walker J, Kazmierski WM et al. Pharmacological disruption of hepatitis C NS5A protein intra- and intermolecular conformations. J Gen Virol 2014; 95:363–372 [View Article][PubMed]
     [Google Scholar]
  49. Chukkapalli V, Berger KL, Kelly SM, Thomas M, Deiters A et al. Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides. Virology 2015; 476:168–179 [View Article][PubMed]
     [Google Scholar]
  50. Tokuriki N, Tawfik DS. Protein dynamism and evolvability. Science 2009; 324:203–207 [View Article][PubMed]
     [Google Scholar]
  51. Haliloglu T, Bahar I. Adaptability of protein structures to enable functional interactions and evolutionary implications. Curr Opin Struct Biol 2015; 35:17–23 [View Article][PubMed]
     [Google Scholar]
  52. Balakrishna AM, Basak S, Manimekalai MS, Grüber G. Crystal structure of subunits D and F in complex gives insight into energy transmission of the eukaryotic V-ATPase from Saccharomyces cerevisiae. J Biol Chem 2015; 290:3183–3196 [View Article][PubMed]
     [Google Scholar]
  53. Dip PV, Kamariah N, Subramanian Manimekalai MS, Nartey W, Balakrishna AM et al. Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli. Acta Crystallogr D Biol Crystallogr 2014; 70:2848–2862 [View Article][PubMed]
     [Google Scholar]
  54. Saw WG, Tria G, Grüber A, Subramanian Manimekalai MS, Zhao Y et al. Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution. Acta Crystallogr D Biol Crystallogr 2015; 71:2309–2327 [View Article][PubMed]
     [Google Scholar]
  55. Konarev PV, Petoukhov MV, Volkov VV, Svergun DI. ATSAS 2.1, a program package for small-angle scattering data analysis. J Appl Crystallogr 2006; 39:277–286 [View Article]
     [Google Scholar]
  56. Svergun DI. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 1992; 25:495–503 [View Article]
     [Google Scholar]
  57. Volkov VV, Svergun DI. Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr 2003; 36:860–864 [View Article]
     [Google Scholar]
  58. Kozin MB, Svergun DI. Automated matching of high- and low-resolution structural models. J Appl Crystallogr 2001; 34:33–41 [View Article]
     [Google Scholar]
  59. Mylonas E, Svergun DI. Accuracy of molecular mass determination of proteins in solution by small-angle X-ray scattering. J Appl Crystallogr 2007; 40:s245–s249 [View Article]
     [Google Scholar]
  60. Rambo RP, Tainer JA. Accurate assessment of mass, models and resolution by small-angle scattering. Nature 2013; 496:477–481 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001000
Loading
Self-association and conformational variation of NS5A domain 1 of hepatitis C virus
J. Gen. Virol. 99, 194 (2018); https://doi.org/10.1099/jgv.0.001000
/content/journal/jgv/10.1099/jgv.0.001000
/content/journal/jgv/10.1099/jgv.0.001000
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error