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 ma mmalian 143B TK- cells to investigate its substrate specificity. The h erpes simplex virus type 1 (HSV-1) TK was similarly expressed for comp arison. Both viral TKs conferred a TK+ phenotype on 143B TK- cells. Th e nucleoside analog ganciclovir (GCV) did not affect the growth of 143 B 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 T K, 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, a nd all had TK activity. In addition, EBV TK was observed to have a thy midylate kinase activity but could not phosphorylate GCV, acyclovir, o r 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 > sta vudine > sorivudine, These results demonstrate that EBV TK substrate s pecificity is narrower than those of alphaherpesvirus TKs and that thy midine analogs may be the most suitable nucleoside antivirals to targe t the enzyme. Clinical implications for gammaherpesviruses are discuss ed,