Purification and characterization of deoxythymidine kinase (dTK) induced in dTK − 3T3 mouse cells by equine herpesvirus type 1 (EHV-1)

Infection of mouse 3T3 cells deficient in cytosol deoxythymidine kinase (dTK −) with the Kentucky A strain of EHV-1 resulted in a 20- to 30-fold increase in cytosol dTK activity. The EHV-1-induced dTK was partially purified from such cells by affinity chromatography on deoxythymidine (dT)-Sepharose and characterized by electrophoretic, enzymatic, and immunological criteria. The purified EHV-1 dTK migrated in polyacrylamide gels with an R f of 0.25 and sedimented in glycerol gradients with an S value of 5.2, corresponding to a molecular weight of 85,000. Deoxycytidine could not serve as an alternative substrate for the viral-induced enzyme. A rabbit antiserum prepared against EHV-1-infected horse cells neutralized the viral-induced dTK activity purified from infected mouse (dTK − 3T3) cells, but not the kinase activities from uninfected 3T3 cells. EHV-1 dTK differed from 3T3 cytosol dTK also in the greater resistance of the viral enzyme to feedback inhibition by dTTP and dCTP, its inhibition by arabinosylthymine, and its ability to utilize a variety of nucleoside triphosphates as phosphate donors. The purified EHV-1 kinase could be distinguished from 3T3 mitochondrial dTK by electrophoretic mobility and inhibition by anti-(EHV-1) serum. These results lend further support to the hypothesis that the dTK induced by EHV-1 is coded by the virus genome.