Selective Abolishment of Pyrimidine Nucleoside Kinase Activity of Herpes Simplex Virus Type 1 Thymidine Kinase by Mutation of Alanine-167 to Tyrosine
- 1Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium (B.D., E.D.C., J.B.); and 2Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom (R.E.)
Abstract
Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that markedly differs from mammalian nucleoside kinases in terms of substrate specificity. It recognizes both pyrimidine 2′-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an attempt to modify this specificity, an HSV-1 TK mutant enzyme containing an alanine-to-tyrosine mutation at amino acid position 167 was constructed. Compared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme was heavily compromised in phosphorylating pyrimidine nucleosides such as (E)-5-(2-bromovinyl)-2′-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV) and lobucavir was only reduced ∼2-fold. Moreover, a markedly decreased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was observed. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene were extremely sensitive to the cytostatic effects of antiherpetic pyrimidine [i.e., (E)-5-(2-bromovinyl)-2′-deoxyuridine] and purine (i.e., GCV) nucleoside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the osteosarcoma cells to a variety of purine nucleoside analogs, whereas there was no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a therapeutic advantage in an in vivo setting due to the markedly reduced dThd competition with GCV for phosphorylation by the HSV-1 TK.
Footnotes
-
Send reprint requests to: Jan Balzarini, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail:jan.balzarini{at}rega.kuleuven.ac.be
-
This work was supported by Project 00/12 from the Flemish “Geconcerteerde Onderzoeksacties,” and the “Belgische Federatie tegen kanker.” Bart Degrève is the recipient of a fellowship from the “Belgische Federatie tegen kanker.”
- Abbreviations:
- TK
- thymidine kinase
- HSV-1
- herpes simplex virus type 1
- GCV
- 9-(1,3-dihydroxy-2-propoxymethyl)guanine (ganciclovir)
- BVDU
- (E)-5-(2-bromovinyl)-2′-deoxyuridine
- BVDU-MP
- 5′-monophosphate of BVDU
- BVaraU
- (E)-5-(2-bromovinyl)-1-β-d-arabinofuranosyluracil
- S-BVDU
- (E)-5-(2-bromovinyl)-2′-deoxy-4′-thiouridine
- araT
- 1-β-d-arabinofuranosylthymine
- LBV
- (R)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine (lobucavir)
- ACV
- 9-(2-hydroxyethoxymethyl)guanine (acyclovir)
- PCV
- 9-[4-hydroxy-3-(hydroxymethyl)but-1-yl]guanine (penciclovir)
- BCV
- (R)-9-(3,4-dihydroxybutyl)guanine (buciclovir)
- VZV
- varicella-zoster virus
- WT
- wild-type
- GFP
- green fluorescent protein
- GST
- glutathione S-transferase
- FACS
- fluorescence-activated cell-sorting analysis
-
- Received June 12, 2000.
- Accepted September 7, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
