Selective abolishment of pyrimidine nucleoside kinase activity of herpes simplex virus type 1 thymidine kinase by mutation of Alanine-167 to tyrosine

Herpes simplex virus type 1 (HSV-1) encodes a thymidine kinase (TK) that ma rkedly differs from mammalian nucleoside kinases in terms of substrate spec ificity. It recognizes both pyrimidine 2'-deoxynucleosides and a variety of purine nucleoside analogs. Based on a computer modeling study and in an at tempt to modify this specificity, an HSV-1 TK mutant enzyme containing an a lanine-to-tyrosine mutation at amino acid position 167 was constructed. Com pared with wild-type HSV-1 TK, the purified mutant HSV-1 TK(A167Y) enzyme w as heavily compromised in phosphorylating pyrimidine nucleosides such as (E )-5-(2-bromovinyl)-2'-deoxyuridine and the natural substrate dThd, whereas its ability to phosphorylate the purine nucleoside analogs ganciclovir (GCV ) and lobucavir was only reduced similar to2-fold. Moreover, a markedly dec reased competition of natural pyrimidine nucleosides (i.e., thymidine) with purine nucleoside analogs for phosphorylation by HSV-1 TK(A167Y) was obser ved. Human osteosarcoma cells transduced with the wild-type HSV-1 TK gene w ere extremely sensitive to the cytostatic effects of antiherpetic pyrimidin e [i.e., (E)-5-(2-bromovinyl)-2'-deoxyuridine] and purine (i.e., GCV) nucle oside analogs. Transduction with the HSV-1 TK(A167Y) gene sensitized the os teosarcoma cells to a variety of purine nucleoside analogs, whereas there w as no measurable cytostatic activity of pyrimidine nucleoside analogs. The unique properties of the A167Y mutant HSV-1 TK may give this enzyme a thera peutic advantage in an in vivo setting due to the markedly reduced dThd com petition with GCV for phosphorylation by the HSV-1 TK.