Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir (original) (raw)

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [ 3 H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S -transferase (GST) fusion protein showed that the enzyme has a comparatively high K m for thymidine (dThd) of ∼33.2 μM. Nearly 50% of the phosphorylated 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 analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2′-deoxyguanosine, 2′-deoxyadenosine, 2′-deoxycytidine, and corresponding analogs did not. Further competition experim...