Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine.

9-(2-Hydroxyethoxymethyl)guanine (acyclo-Guo), is an effective and selective inhibitor of herpes simplex virus replication. This purine nucleoside analogue was found to be phosphorylated at a 30to 120-fold faster rate with extracts of Vero cells infected with herpes simplex virus than with extracts of uninfected cells. The enzyme responsible for this reaction was identified as the virus-coded thymidine (dThd) kinase (EC 2.7.1.75). Evidence for this included: 1) the proportionate increase in the rates of phosphorylation of dThd (measured at pH 6) and acyclo-Guo after infection of Vero cells, 2) the concomitant loss of both activities in mutant strains of viruses deficient in dThd kinase, 3) the co-migration of the two activities on polyacrylamide gel electrophoresis, 4) the co-purification of the two activities with an affinity chromatography procedure that separates viral from host cell dThd kinase, and 5) the similar inhibitor specificities when either compound was used as the radioactive substrate. The amount of extractable kinase has been calculated to be sufficient to account for the amount of phosphorylated acyclo-Guo found in treated infected cells in tissue culture. Low acyclo-Guo phosphorylating levels in cells infected with either of two herpes simplex virus mutants or with vaccinia virus correlated with low effectiveness of the compound against the virus in tissue culture. Relative rates of phosphorylation with nonradioactive nucleoside analogues were determined by a method employing [14C]phosphoenolpyruvate. AcycloGuo was phosphorylated at a faster rate than any other purine derivative tested. Other effective antiviral purine derivatives were not phosphorylated by the kinase and thus must be activated by a different mechanism.