Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to enco de a protein similar to the thymidine kinase (TK) enzyme of other herpesvir uses. Expressed in mammalian cells, ORF 21 was found to have low TK activit y, based on poor growth in media containing hypoxanthine-aminopterin-thymid ine (HAT) and low incorporation of [H-3] thymidine into high-molecular-weig ht DNA, Kinetic analysis using HHV8 TK as a purified glutathione S-transfer ase (GST) fusion protein showed that the enzyme has a comparatively high K- m for thymidine (dThd) of similar to 33.2 mu M. Nearly 50% of the phosphory lated product of the reaction with dThd was thymidylate, This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate, Thymidine analog s competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxygu anosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not, Further competition experiments revealed that the nucleoside analog g anciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK- cells expressing HHV8 TK phosphorylated GCV very poorly and were not s usceptible to GCV toxicity compared to parental cells, Phosphorylation of [ H-3] GCV by a purified GST-HHV8 TK fusion protein was not detected by high- pressure liquid chromatography analysis. Structural features of HHV8 TK sub strate recognition were investigated. Therapeutic implications of these fin dings are discussed.