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Journal of Virology, January 2000, p. 684-692, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Erik A. Gustafson,1,2 Raymond F. Schinazi,3 and Joyce D. Fingeroth1,2,*

Divisions of Infectious Disease and Experimental Medicine, Beth Israel Deaconess Medical Center,1 and Harvard Medical School,2 Boston, Massachusetts 02115, and Veterans Affairs Medical Center, Emory University, Decatur, Georgia 300333

Received 15 July 1999/Accepted 14 October 1999

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 [3H]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 Km 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 experiments revealed that the nucleoside analog ganciclovir (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 susceptible to GCV toxicity compared to parental cells. Phosphorylation of [3H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.

* Corresponding author. Mailing address: Divisions of Infectious Disease and Experimental Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115. Phone: (617) 667-0072. Fax: (617) 975-5243. E-mail: jfingero{at}caregroup.harvard.edu.

Journal of Virology, January 2000, p. 684-692, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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