Anti-Epstein-Barr virus (EBV) activity of beta-L-5-iododioxolane uracil is dependent on EBV thymidine kinase

Abstract

beta-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration = 0.03 microM) with low cytotoxicity (50% cytotoxic concentration = 1,000 microM). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate beta-L-5-iododioxolane uracil to the monophosphate metabolite. The K(m) of beta-L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 microM, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2' fluoro-5-methyl-beta-L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V(max) is seven times higher than that of thymidine. The anti-EBV activity of beta-L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5'-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that beta-L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug.

FIG. 1

Chemical structure of L-I-OddU and EBV inhibitor 5′-Et-dThd.

FIG. 2

(A) Detection of linear and circular EBV DNA in cells with or without L-I-OddU and L-Br-OddU. Lanes: 1, H1 cells with no treatment; 2, H1 cells plus dimethyl sulfoxide (DMSO); 3, H1 cells plus 1 μM L-I-OddU; 4, H1 cells plus 1 μM L-Br-OddU; 5, L5 cells with no treatment; 6, L5 cells plus DMSO; 7, L5 cells plus 1 μM L-I-OddU; 8, L5 cells plus 1 μM L-Br-OddU; 9, CEM cells with no treatment (CEM cells are human T cells which do not contain any EBV genome). (B) Western blotting by anti-EBV TK antibody. Lanes: 1, H1 cells with no treatment; 2, H1 cells plus DMSO; 3, H1 cells plus 1 μM L-I-OddU; 4, H1 cells plus 1 μM L-Br-OddU; 5, L5 cells with no treatment; 6, L5 cells plus DMSO; 7, L5 cells plus 1 μM L-I-OddU; 8, L5 cells plus 1 μM L-Br-OddU; 9, CEM cells with no treatment. The TK protein is marked at 69 kDa and a human protein (HP) that has bound nonspecifically to this antibody serves as a load control.

FIG. 3

(A) Effect of 5′-Et-dThd on thymidine phosphorylation by EBV (), human cytosolic TK (○), and mitochondrial dPydK (▴). The concentration of cold dThd in the reaction mix was 20 μM. (B) Lineweaver-Burk plots of various L-I-OddU concentrations with or without 5′-Et-dThd (large figure) and the replot (left-side inset). The K_i value of 5′-Et-dThd obtained was 4 μM. The 5′-Et-dThd concentrations tested were 0 μM (●), 7.5 μM (▿), 15 μM (+), 30 μM (▴), and 60 μM (▪).

FIG. 4

Effect of 5′-Et-dThd on the anti-EBV activity of L-I-OddU. The virus amount produced without L-I-OddU treatment was used as the 100% level. The 5′-Et-dThd concentrations tested were 0 μM (▿), 0.8 μM (▴), 4 μM (○), and 20 μM ().