Skip to main content

Advertisement

SpringerLink
Log in

Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

Feline herpesvirus-1 (FHV-1) causes a severe upper respiratory and ocular disease in cats. An effective antiviral compound is required for treating FHV-1 infections. The virus-encoded thymidine kinase (TK) is the molecular basis for selective activation of commonly used antiviral nucleoside analogue drugs, e.g. acyclovir (ACV), penciclovir (PCV) and ganciclovir (GCV). The substrate specificity of a recombinant FHV-1 TK, expressed in Escherichia coli, was studied. FHV-1 TK efficiently phosphorylated its natural substrate deoxythymidine. However, it exhibited relatively lower affinity for the guanosine analogue substrates. PCV was most efficiently phosphorylated, followed by GCV, with approximately twofold reduction in the phosphorylation rate. The lowest phosphorylation rate was recorded for ACV. To correlate these biochemical data with structural features of the FHV-1 TK, a three-dimensional (3D) model of this enzyme was constructed based on sequence homology with two other herpesviral TKs, encoded by equine herpesvirus-4 (EHV-4) and herpes simplex-1 (HSV-1). Mutational analysis of the amino acids forming the FHV-1 TK active site identified two residues (Y29 and F144) as being critical for the differential ability of this enzyme to phosphorylate nucleoside analogues. A double substitution of Y29H/F144Y resulted in a threefold increase in the ACV phosphorylation rate.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Alber F, Kuonen O, Scapozza L, Folkers G, Carloni P (1998) Density functional studies on herpes simplex virus type 1 thymidine kinase–substrate interactions: the role of Tyr-172 and Met-128 in thymine fixation. Proteins 31:453–459

    Article  PubMed  CAS  Google Scholar 

  2. Andrew SE (2001) Ocular manifestations of feline herpesvirus. J Feline Med Surg 3:9–16

    Article  PubMed  CAS  Google Scholar 

  3. Baker D, Sali A (2001) Protein structure prediction and structural genomics. Science 294:93–96

    Article  PubMed  CAS  Google Scholar 

  4. Bennett MS, Wien F, Champness JN, Batuwangala T, Rutherford T, Summers WC, Sun H, Wright G, Sanderson MR (1999) Structure to 1.9 A resolution of a complex with herpes simplex virus type-1 thymidine kinase of a novel, non-substrate inhibitor: X-ray crystallographic comparison with binding of aciclovir. FEBS Lett 443:121–125

    Article  PubMed  CAS  Google Scholar 

  5. Blundell TL, Jhoti H, Abell C (2002) High-throughput crystallography for lead discovery in drug design. Nat Rev Drug Discov 1:45–54

    Article  PubMed  CAS  Google Scholar 

  6. Brooks BR, Bruccolesi RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy minimisation and dynamics calculations. J Comput Chem 4:187–217

    Article  CAS  Google Scholar 

  7. Brown DG, Visse R, Sandhu G, Davies A, Rizkallah PJ, Melitz C, Summers WC, Sanderson MR (1995) Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nat Struct Biol 2:876–881

    Article  PubMed  CAS  Google Scholar 

  8. Champness JN, Bennett MS, Wien F, Visse R, Summers WC, Herdewijn P, de Clerq E, Ostrowski T, Jarvest RL, Sanderson MR (1998) Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands. Proteins 32:350–361

    Article  PubMed  CAS  Google Scholar 

  9. Collins P (1983) The spectrum of antiviral activities of acyclovir in vitro and in vivo. J Antimicrob Chemother 12(Suppl B):19–27

    PubMed  CAS  Google Scholar 

  10. Copeland RA (2000) Enzymes: a practical introduction to structure, mechanism and data analysis. 2nd edn. Wiley, New York

    Google Scholar 

  11. Darby G (1994) A history of antiherpes research. Antivir Chem Chemother 5:3–9

    CAS  Google Scholar 

  12. De Clercq E, Brancale A, Hodge AV, Field HJ (2006) Antiviral chemistry and chemotherapy’s current antiviral agents FactFile 2006 (1st edition). Antivir Chem Chemother 17:113–166

    PubMed  Google Scholar 

  13. Earnshaw DL, Bacon TH, Darlison SJ, Edmonds K, Perkins RM, Vere Hodge RA (1992) Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. Antimicrob Agents Chemother 36:2747–2757

    PubMed  CAS  Google Scholar 

  14. Elion GB (1993) Acyclovir: discovery, mechanism of action, and selectivity. J Med Virol (Suppl 1):2–6

  15. Evans JS, Lock KP, Levine BA, Champness JN, Sanderson MR, Summers WC, McLeish PJ, Buchan A (1998) Herpesviral thymidine kinases: laxity and resistance by design. J Gen Virol 79(Pt 9):2083–2092

    PubMed  CAS  Google Scholar 

  16. Fersht A (1999) The basic equations of enzyme kinetics structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. W. H. Freeman and Company, New York, pp 103–129

    Google Scholar 

  17. Fetzer J, Michael M, Bohner T, Hofbauer R, Folkers G (1994) A fast method for obtaining highly pure recombinant herpes simplex virus type 1 thymidine kinase. Protein Expr Purif 5:432–441

    Article  PubMed  CAS  Google Scholar 

  18. Field HJ, Whitley RJ (2005) Antiviral chemotherapy. In: Mahy BWJ, Meulen V (eds) Topley and Wilson’s microbiology and microbial infections, vol 2, 10th edn. Hodder Arnold, London, Virology, pp 1605–1645

  19. Fiser A (2004) Protein structure modeling in the proteomics era. Expert Rev Proteomics 1:97–110

    Article  PubMed  CAS  Google Scholar 

  20. Furman PA, St Clair MH, Spector T (1984) Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase. J Biol Chem 259:9575–9579

    PubMed  CAS  Google Scholar 

  21. Fyfe JA, McKee SA, Keller PM (1983) Altered thymidine–thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2′-deoxyuridine. Mol Pharmacol 24:316–323

    PubMed  CAS  Google Scholar 

  22. Galle LE (2004) Antiviral therapy for ocular viral disease. Vet Clin North Am Small Anim Pract 34:639–653

    Article  PubMed  Google Scholar 

  23. Gardberg A, Shuvalova L, Monnerjahn C, Konrad M, Lavie A (2003) Structural basis for the dual thymidine and thymidylate kinase activity of herpes thymidine kinases. Structure (Camb) 11:1265–1277

    Article  CAS  Google Scholar 

  24. Gaskell R, Willoughby K (1999) Herpesviruses of carnivores. Vet Microbiol 69:73–88

    Article  PubMed  CAS  Google Scholar 

  25. Gaskell RM, Dawson S (1994) Chapter 14: Viral-induced upper respiratory tract disease. In: Chandler EA, Gaskell CJ, Gaskell RM (eds) Feline medicine and therapeutics. Blackwell, Oxford, pp 453–472

    Google Scholar 

  26. Gentry GA (1992) Viral thymidine kinases and their relatives. Pharmacol Ther 54:319–355

    Article  PubMed  CAS  Google Scholar 

  27. Gerber S, Folkers G (1996) A new method for quantitative determination of tritium-labeled nucleoside kinase products adsorbed on DEAE-cellulose. Biochem Biophys Res Commun 225:263–267

    Article  PubMed  CAS  Google Scholar 

  28. Ginalski K (2006) Comparative modeling for protein structure prediction. Curr Opin Struct Biol 16:172–177

    Article  PubMed  CAS  Google Scholar 

  29. Griffiths PD (1994) Spectrum of activity of antiherpesvirus drugs. Antivir Chem Chemother 5:17–22

    CAS  Google Scholar 

  30. Gustafson EA, Chillemi AC, Sage DR, Fingeroth JD (1998) The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase. Antimicrob Agents Chemother 42:2923–2931

    PubMed  CAS  Google Scholar 

  31. Gustafson EA, Schinazi RF, Fingeroth JD (2000) Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir. J Virol 74:684–692

    Article  PubMed  CAS  Google Scholar 

  32. Harrison PT, Thompson R, Davison AJ (1991) Evolution of herpesvirus thymidine kinases from cellular deoxycytidine kinase. J Gen Virol 72( Pt 10): 2583–2586

    PubMed  CAS  Google Scholar 

  33. Hillisch A, Pineda LF, Hilgenfeld R (2004) Utility of homology models in the drug discovery process. Drug Discov Today 9:659–669

    Article  PubMed  CAS  Google Scholar 

  34. Hooft RW, Sander C, Vriend G (1997) Objectively judging the quality of a protein structure from a Ramachandran plot. Comput Appl Biosci 13:425–430

    PubMed  CAS  Google Scholar 

  35. Hussein ITM, Menashy RV, Field HJ (2007) Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase. Antiviral Res (in press)

  36. Kit S, Ichimura H, De Clercq E (1987) Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase. Antiviral Res 7:53–67

    Article  PubMed  CAS  Google Scholar 

  37. Larder BA, Cheng YC, Darby G (1983) Characterization of abnormal thymidine kinases induced by drug-resistant strains of herpes simplex virus type 1. J Gen Virol 64(Pt 3):523–532

    PubMed  CAS  Google Scholar 

  38. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK––a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    Article  CAS  Google Scholar 

  39. Lock MJ, Thorley N, Teo J, Emery VC (2002) Azidodeoxythymidine and didehydrodeoxythymidine as inhibitors and substrates of the human herpesvirus 8 thymidine kinase. J Antimicrob Chemother 49:359–366

    Article  PubMed  CAS  Google Scholar 

  40. Loutsch JM, Sainz B Jr, Marquart ME, Zheng X, Kesavan P, Higaki S, Hill JM, Tal-Singer R (2001) Effect of famciclovir on herpes simplex virus type 1 corneal disease and establishment of latency in rabbits. Antimicrob Agents Chemother 45:2044–2053

    Article  PubMed  CAS  Google Scholar 

  41. Luthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85

    Article  PubMed  CAS  Google Scholar 

  42. Maggs DJ, Clarke HE (2004) In vitro efficacy of ganciclovir, cidofovir, penciclovir, foscarnet, idoxuridine, and acyclovir against feline herpesvirus type-1. Am J Vet Res 65:399–403

    Article  PubMed  CAS  Google Scholar 

  43. Maggs DJ (2005) Update on pathogenesis, diagnosis, and treatment of feline herpesvirus type 1. Clin Tech Small Anim Pract 20:94–101

    Article  PubMed  Google Scholar 

  44. Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A (2000) Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325

    Article  PubMed  CAS  Google Scholar 

  45. McDonald IK, Thornton JM (1994) Satisfying hydrogen bonding potential in proteins. J Mol Biol 238:777–793

    Article  PubMed  CAS  Google Scholar 

  46. Miller WH, Miller RL (1982) Phosphorylation of acyclovir diphosphate by cellular enzymes. Biochem Pharmacol 31:3879–3884

    Article  PubMed  CAS  Google Scholar 

  47. Nasisse MP, Guy JS, Davidson MG, Sussman W, De Clercq E (1989) In vitro susceptibility of feline herpesvirus-1 to vidarabine, idoxuridine, trifluridine, acyclovir, or bromovinyldeoxyuridine. Am J Vet Res 50:158–160

    PubMed  CAS  Google Scholar 

  48. Nasisse MP, Dorman DC, Jamison KC, Weigler BJ, Hawkins EC, Stevens JB (1997) Effects of valacyclovir in cats infected with feline herpesvirus 1. Am J Vet Res 58:1141–1144

    PubMed  CAS  Google Scholar 

  49. Owens JG, Nasisse MP, Tadepalli SM, Dorman DC (1996) Pharmacokinetics of acyclovir in the cat. J Vet Pharmacol Ther 19:488–490

    PubMed  CAS  Google Scholar 

  50. Pue MA, Benet LZ (1993) Pharmacokinetics of famciclovir in man. Antivir Chem Chemother 4:47–55

    CAS  Google Scholar 

  51. Roberts GB, Fyfe JA, McKee SA, Rahim SG, Daluge SM, Almond MR, Rideout JL, Koszalka GW, Krenitsky TA (1993) Varicella-zoster virus thymidine kinase. Characterization and substrate specificity. Biochem Pharmacol 46:2209–2218

    Article  PubMed  CAS  Google Scholar 

  52. Robertson GR, Whalley JM (1988) Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase. Nucleic Acids Res 16:11303–11317

    Article  PubMed  CAS  Google Scholar 

  53. Roizman B, Pellett PE (2001) Herpesviridae. In: Knipe DM, Howley PM (eds) Fields virology. Lippincott Williams and Wilkins, Philadelphia, pp 2381–2397

    Google Scholar 

  54. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815

    Article  PubMed  CAS  Google Scholar 

  55. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  56. Shi J, Blundell TL, Mizuguchi K (2001) FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol 310:243–257

    Article  PubMed  CAS  Google Scholar 

  57. Solaroli N, Johansson M, Balzarini J, Karlsson A (2006) Substrate specificity of three viral thymidine kinases (TK): vaccinia virus TK, feline herpesvirus TK, and canine herpesvirus TK. Nucleosides Nucleotides Nucleic Acids 25:1189–1192

    Article  PubMed  CAS  Google Scholar 

  58. Stiles J (2003) Feline herpesvirus. Clin Tech Small Anim Pract 18:178–185

    Article  PubMed  Google Scholar 

  59. Vere Hodge RA, Perkins RM (1989) Mode of action of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against herpes simplex virus in MRC-5 cells. Antimicrob Agents Chemother 33:223–229

    Google Scholar 

  60. Wild K, Bohner T, Folkers G, Schulz GE (1997) The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue. Protein Sci 6:2097–2106

    Article  PubMed  CAS  Google Scholar 

  61. Williams DL, Robinson JC, Lay E, Field H (2005) Efficacy of topical aciclovir for the treatment of feline herpetic keratitis: results of a prospective clinical trial and data from in vitro investigations. Vet Rec 157:254–257

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by a generous research grant from Gilead Sciences. ITMH was funded by a BP Cambridge PhD scholarship.

Author information

Author notes

  1. Islam T. M. Hussein

    Present address: HIV Drug Resistance Program, NIH/NCI-Frederick, Frederick, MD, 21702, USA

Authors and Affiliations

  1. Department of Veterinary Medicine, Cambridge University, Cambridge, UK

    Islam T. M. Hussein, Laurence S. Tiley & Hugh J. Field

  2. Department of Biochemistry, Cambridge University, Cambridge, UK

    Ricardo Núñez Miguel

Authors
  1. Islam T. M. Hussein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ricardo Núñez Miguel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laurence S. Tiley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hugh J. Field
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hugh J. Field.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hussein, I.T.M., Miguel, R.N., Tiley, L.S. et al. Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase. Arch Virol 153, 495–505 (2008). https://doi.org/10.1007/s00705-007-0021-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-007-0021-6

Keywords

Search

Navigation