Studies were undertaken to determine the enzymatic basis for the selective inhibition of herpes simplex virus (HSV) by 5-halogenated analogues of deoxycytidine (dCyd). The following results suggest that HSV-2-induced thymidine (dThd), dCyd, and 5-bromodeoxycytidine (5-Br-dCyd) kinase are catalytic activities of the same protein: (a) cofractionation of the virus-induced activities was demonstrated during ammonium sulfate fractionation, Sephadex gel filtration, DEAE ion-exchange chromatography, and polyacrylamide disc gel electrophoresis; (b) the three activities were inactivated at the same rate by heat and were equally protected from thermal inactivation in the presence of dThd, dCyd, or 5-Br-dCyd; (c) the three activities were similarly inhibited by dTTP but were not affected by dCTP; (d) the subunit aggregation of the protein which catalyzes the three reactions was affected identically by MgCl2 and ATP; and (e) viral mutants selected for the absence of one activity (either dThd or dCyd kinase) were deficient in the induction of all three activities. Kinetic studies suggested that phosphorylation of the three nucleosides occurs at a common active site. Substrate specificity studies with the HSV-2 pyrimidine nucleoside kinase indicated that, generally, the 5-substituted analogues of deoxyuridine were more readily phosphorylated than the 5-substituted analogues of dCyd. 5-Methyl and 5-halogenated pyrimidine ribonucleosides are effective substrates for the enzyme, Phosphorylation of purine nucleosides was not detected. The activities of various nucleoside triphosphates as phosphate donor for dThd kinase were in order of CTP > dCTP > dATP > ATP > UTP > GTP; for dCyd kinase, dATP > ATP > CTP > CTP > UTP; for 5-Br-dCyd kinase, CTP > dATP > ATP > dCTP > UTP. ADP, AMP, dGTP, dTTP, dUTP, dCMP, and dTMP were not active as phosphate donors. Experiments with kinase-deficient mutants suggested that the antiviral activity of 5-Br-dCyd is dependent upon the induction of a functional pyrimidine nucleoside kinase. Furthermore, kinetic studies indicated that in contrast to its relatively poor affinity for the host cell cytosol kinases, 5-Br-dCyd has a relatively high affinity for the HSV-2 induced kinase (Km = 1.3 μM). Therefore, the basis for the selective inhibition of HSV-2 by 5-halogenated analogues of dCyd is reflected in the induction of a pyrimidine nucleoside kinase with a high affinity for 5-Br-dCyd.
ASJC Scopus subject areas