Alterations in substrate specificity and physicochemical properties of deoxythymidine kinase of a drug-resistant herpes simplex virus type 1 mutant.
The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).
D001087:Arabinonucleosides / Q000494:pharmacology; D001769:Blood; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives / Q000494:pharmacology; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D004790:Enzyme Induction; D006358:Hot Temperature; D009154:Mutation; D010766:Phosphorylation; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013937:Thymidine Kinase / Q000378:metabolism*
D001087:Arabinonucleosides; C020235:brivudine; C024722:5-methoxymethyl-2'-deoxyuridine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; C010032:thymine arabinoside; D013936:Thymidine; D003857:Deoxyuridine