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

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK(−) cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK(+) phenotype on 143B TK(−) cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK(−) cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N(−), a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2′-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.