DIFFERENTIAL PHOSPHORYLATION OF (E)-5-(2-BROMOVINYL)-2'-DEOXYURIDINE MONOPHOSPHATE BY THYMIDYLATE KINASES FROM HERPES-SIMPLEX VIRUSES TYPE-1 AND TYPE-2 AND VARICELLA ZOSTER VIRUS

Abstract

5-(2-Bromovinyl)-2''-deoxyuridine (BrVdUrd) is a potent and selective inhibitor of herpes simplex type 1 (HS-1) and varicella zoster (VZ) virus replication but is much less potent against HS-2 virus replication. A possible enzymatic basis for this difference is reported here to involve the virus-coded dThd-dTMP kinases (EC 2.7.1.21) from the 3 virus strains. The thymidine kinases from the 3 virus strains were purified by affinity chromatography. In addition to catalyzing the phosphorylation of nucleosides, each of the 3 purified enzymes catalyzed the phosphorylation of thymidylate to its diphosphate but at strikingly different rates. The relative amounts of extractable virus-coded thymidylate kinases were estimated to be 100/2 per 40 for mouse fibroblast LMTK-, African green monkey kidney vero and human fibroblast WI-38 cells infected with HS-1, HS-2 and VZ viruses, respectively. Extracts of cells infected with HS-1 virus catalyzed the phosphorylation of the monophosphate of BrVdUrd to its diphosphate. The product was not detected with extracts from cells infected with HS-2 virus. The ratios of rates with 0.5 mM BrVdUrd monophosphate vs. 0.1 mM dTMP as substrates for each of the purified dThd-dTMP kinases from HS-1, HS-2 and VZ viruses and the dTMP kinase from host cells were, respectively, 0.09, < 0.002, 0.03 and < 0.0002. These observations correlated with the relative sensitivities of these viruses to BrVdUrd in cell culture and suggest that, if BrVdUrd exerts its effect as a triphosphate, the inefficient phosphorylation of the monophosphate contributes to the insensitivity of HS-2 virus to this agent.