Virus Genes

, Volume 55, Issue 2, pp 227–232 | Cite as

Recombinant HCV NS3 and NS5B enzymes exhibit multiple posttranslational modifications for potential regulation

  • Sergio Hernández
  • Ariel Díaz
  • Alejandra Loyola
  • Rodrigo A. VillanuevaEmail author


Posttranslational modification (PTM) of proteins is critical to modulate protein function and to improve the functional diversity of polypeptides. In this report, we have analyzed the PTM of both hepatitis C virus NS3 and NS5B enzyme proteins, upon their individual expression in insect cells under the baculovirus expression system. Using mass spectrometry, we present evidence that these recombinant proteins exhibit diverse covalent modifications on certain amino acid side chains, such as phosphorylation, ubiquitination, and acetylation. Although the functional implications of these PTM must be further addressed, these data may prove useful toward the understanding of the complex regulation of these key viral enzymes and to uncover novel potential targets for antiviral design.


Hepatitis C virus HCV NS3 NS5B Posttranslational modification Protein regulation 



We thank Drs. Stephen Barnes and Landon Wilson from TMPL at UAB for the mass spectrometry analyses. We appreciate continuous support from Dr. Stanley M. Lemon, and Dr. Minkyung Yi. We thank Dr. Takaji Wakita for the transfer of pFGR-JFH1 plasmid utilized in our research. We thank all members of our laboratories for fruitful discussions while this work was carried out. This research has been supported by grants from CONICYT, Basal Project AFB 170004 (A.L.), FONDECYT 1160480 (A.L.), FONDECYT 1100200 (R.A.V.), and PCHA/Doctorado Nacional/2014-21140956 (S.H.).

Author’s contributions

RAV contributed to the study conception and design. SH and AD performed the experiments. RAV wrote the manuscript. SH, AD, RAV, and AL checked and revised it. RAV and AL contributed with funding. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals that required ethical approval.

Supplementary material

11262_2019_1638_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1847 KB)


  1. 1.
    Tellinghuisen T, Evans M, Hahn T, You S, Rice C (2007) Studying hepatitis C virus: making the best of a bad virus. J Virol 81:8853–8867CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Paul D, Madan V, Bartenschlager R (2014) Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host Microbe 16(5):569–579CrossRefPubMedGoogle Scholar
  3. 3.
    Villanueva RA, Rouille Y, Dubuisson J (2005) Interactions between virus proteins and host cell membranes during the viral life cycle. Int Rev Cytol 245:171–244CrossRefPubMedGoogle Scholar
  4. 4.
    Wang H, Tai AW (2016) Mechanisms of cellular membrane reorganization to support hepatitis C virus replication. Viruses 8(5):E142CrossRefPubMedGoogle Scholar
  5. 5.
    Yao N, Reichert P, Taremi S, Prosise W, Weber P (1999) Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase. Structure 7:1353–1363CrossRefPubMedGoogle Scholar
  6. 6.
    Wölk B, Sansonno D, Kräusslich H, Dammacco F, Rice C, Blum H, Moradpour D (2000) Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines. J Virol 74:2293–2304CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Frick DN, Rypma RS, Lam AMI, Gu B (2004) The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently. J Biol Chem 279(2):1269–1280CrossRefPubMedGoogle Scholar
  8. 8.
    Pang PS, Jankowsky E, Planet PJ, Pyle AM (2002) The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21(5):1168–1176CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kolykhalov A, Mihalik K, Feinstone S, Rice C (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 74:2046–2051CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lam A, Frick D (2006) Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase. J Virol 80:404–411CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bressanelli S, Tomei L, Roussel A, Incitti I, Vitale R, Mathieu M, Francesco RD, Rey F (1999) Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc Natl Acad Sci USA 96:13034–13039CrossRefPubMedGoogle Scholar
  12. 12.
    Sesmero E, Thorpe IF (2015) Using the hepatitis C virus RNA-dependent RNA polymerase as a model to understand viral polymerase structure, function and dynamics. Viruses 7(7):3974–3994CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Bressanelli S, Tomei L, Rey F, Francesco RD (2002) Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J Virol 76:3482–3492CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Biswal B, Cherney M, Wang M, Chan L, Yannopoulos C, Bilimoria D, Nicolas O, Bedard J, James M (2005) Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J Biol Chem 280:18202–18210CrossRefPubMedGoogle Scholar
  15. 15.
    Tomei L, Vitale R, Incitti I, Serafini S, Altamura S, Vitelli A, Francesco RD (2000) Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence. J Gen Virol 81:759–767CrossRefPubMedGoogle Scholar
  16. 16.
    Schmidt-Mende J, Bieck E, Hugle T, Penin F, Rice C, Blum H, Moradpour D (2001) Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276:44052–44063CrossRefPubMedGoogle Scholar
  17. 17.
    Shih C, Chen C, Chen S, Lee Y (1995) Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation. J Virol 69:1160–1171PubMedPubMedCentralGoogle Scholar
  18. 18.
    Franck N, Seyec JL, Guguen-Guillouzo C, Erdtmann L (2005) Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome. J Virol 79:2700–2708CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hundt J, Li Z, Liu Q (2013) Post-translational modifications of hepatitis C viral proteins and their biological significance. World J Gastroenterol 19(47):8929–8939CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Rho J, Choi S, Seong Y, Choi J, Im D (2001) The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1. J Virol 75:8031–8044CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Liefhebber J, Hensbergen P, Deelder A, Spaan W, Leeuwen H (2010) Characterization of hepatitis C virus NS3 modifications in the context of replication. J Gen Virol 91:1013–1018CrossRefPubMedGoogle Scholar
  22. 22.
    Yu G, Lee K, Gao L, Lai M (2006) Palmitoylation and polymerization of hepatitis C virus NS4B protein. J Virol 80:6013–6023CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Huang Y, Staschke K, Francesco RD, Tan S (2007) Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Virology 364:1–9CrossRefPubMedGoogle Scholar
  24. 24.
    Ross-Thriepland D, Harris M (2015) Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on! J Gen Virol 96(4):727–738CrossRefPubMedGoogle Scholar
  25. 25.
    Hwang S, Park K, Kim Y, Sung Y, Lai M (1997) Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology 227:439–446CrossRefPubMedGoogle Scholar
  26. 26.
    Han SH, Kim SJ, Kim EJ, Kim TE, Moon JS, Kim GW, Lee SH, Cho K, Yoo JS, Son WS, Rhee JK, Han SH, Oh JW (2014) Phosphorylation of hepatitis C virus RNA polymerases ser29 and ser42 by protein kinase C related kinase 2 regulates viral RNA replication. J Virol 88(19):11240–11252CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kim S, Kim J, Kim Y, Lim H, Oh J (2004) Protein kinase C-related kinase 2 regulates hepatitis C virus RNA polymerase function by phosphorylation. J Biol Chem 279:50031–50041CrossRefPubMedGoogle Scholar
  28. 28.
    Hernández S, Figueroa D, Correa S, Díaz A, Aguayo D, Villanueva RA (2015) Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase. Biochem Biophys Res Commun 466(1):21–27CrossRefPubMedGoogle Scholar
  29. 29.
    Jakubiec A, Jupin I (2007) Regulation of positive-strand RNA virus replication: the emerging role of phosphorylation. Virus Res 129:73–79CrossRefPubMedGoogle Scholar
  30. 30.
    Kuang W, Lin Y, Jean F, Huang Y, Tai C, Chen D, Chen P, Hwang L (2004) Hepatitis C virus NS3 RNA helicase activity is modulated by the two domains of NS3 and NS4A. Biochem Biophys Res Commun 317:211–217CrossRefPubMedGoogle Scholar
  31. 31.
    Welbourn S, Pause A (2007) The hepatitis C virus NS2/3 protease. Curr Issues Mol Biol 9:63–69PubMedGoogle Scholar
  32. 32.
    Hochstrasser M (2009) Origin and function of ubiquitin-like proteins. Nature 458:422–429CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Fiore PD, Polo S, Hofmann K (2003) When ubiquitin meets ubiquitin receptors: a signalling connection. Nat Rev Mol Cell Biol 4:491–497CrossRefPubMedGoogle Scholar
  34. 34.
    Schnell J, Hicke L (2003) Non-traditional functions of ubiquitin and ubiquitin-binding proteins. J Biol Chem 278:35857–35860CrossRefPubMedGoogle Scholar
  35. 35.
    Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315:201–205CrossRefPubMedGoogle Scholar
  36. 36.
    Leestemaker Y, Ovaa H (2017) Tools to investigate the ubiquitin proteasome system. Drug Discov Today Technol 26:25–31CrossRefPubMedGoogle Scholar
  37. 37.
    Dwane L, Gallagher WM, Ní Chonghaile T, O’Connor DP (2017) The emerging role of non-traditional ubiquitination in oncogenic pathways. J Biol Chem 292(9):3543–3551CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Haqshenas G (2012) The conserved lysine 151 of HCV NS5B modulates viral genome replication and infectious virus production. J Viral Hepat 19:862–866CrossRefPubMedGoogle Scholar
  39. 39.
    Spange S, Wagner T, Heinzel T, Krämer O (2009) Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 41:185–198CrossRefPubMedGoogle Scholar
  40. 40.
    Buuh ZY, Lyu Z, Wang RE (2018) Interrogating the roles of post-translational modifications of non-histone proteins. J Med Chem 61(8):3239–3252CrossRefPubMedGoogle Scholar
  41. 41.
    Spange S, Wagner T, Heinzel T, Krämer OH (2009) Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 41(1):185–198CrossRefPubMedGoogle Scholar
  42. 42.
    Reissner K, Aswad D (2003) Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cell Mol Life Sci 60:1281–1295CrossRefPubMedGoogle Scholar
  43. 43.
    Legrand P, Rioux V (2010) The complex and important cellular and metabolic functions of saturated fatty acids. Lipids 45:941–946CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Aicart-Ramos C, Valero R, Rodriguez-Crespo I (2011) Protein palmitoylation and subcellular trafficking. Biochim Biophys Acta 1808:2981–2994CrossRefPubMedGoogle Scholar
  45. 45.
    Nishi Y, Yoh J, Hiejima H, Kojima M (2011) Structures and molecular forms of the ghrelin-family peptides. Peptides 32:2175–2182CrossRefPubMedGoogle Scholar
  46. 46.
    Schwandt SE1, Peddu SC, Riley LG (2010) Differential roles for octanoylated and decanoylated ghrelins in regulating appetite and metabolism. Int J Pept 2010:275804CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Riley LG (2013) Different forms of ghrelin exhibit distinct biological roles in tilapia. Front Endocrinol (Lausanne) 4:118CrossRefGoogle Scholar
  48. 48.
    Khatib N, Gaidhane S, Gaidhane AM, Khatib M, Simkhada P, Gode D, Zahiruddin QS (2014) Ghrelin: ghrelin as a regulatory peptide in growth hormone secretion. J Clin Diagn Res 8(8):MC13–M7PubMedPubMedCentralGoogle Scholar
  49. 49.
    Kapadia S, Chisari F (2005) Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci USA 102:2561–2566CrossRefPubMedGoogle Scholar
  50. 50.
    Kost T, Condreay J, Jarvis D (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Kollewe C, Vilcinskas A (2013) Production of recombinant proteins in insect cells. Am J Biochem Biotechnol 9:255–271CrossRefGoogle Scholar
  52. 52.
    van Oers M, Pijlman G, Vlak J (2015) Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology. J Gen Virol 96:6–23CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sergio Hernández
    • 1
    • 2
  • Ariel Díaz
    • 1
  • Alejandra Loyola
    • 1
  • Rodrigo A. Villanueva
    • 1
    Email author
  1. 1.Fundación Ciencia &, VidaÑuñoaChile
  2. 2.Architecture et Fonction des Macromolécules Biologiques, CNRS UMR7257, Department of Medicinal ChemistryAix Marseille UniversiteMarseilleFrance

Personalised recommendations