Substrate specificity of Epstein-Barr virus thymidine kinase

Abstract

Purified recombinant protein encoded by the BXLF-I open reading frame of the Epstein-Barr virus genome has thymidine kinase activity. The substrate behaviors of various nucleosides toward this enzyme were tested. Halogenated deoxyuridines, zidovudine, and bromovinyldeoxyuridine are efficient substrates, while acyclovir and dihydroxypropylmethylguanine are relatively poor substrates for the Epstein-Barr virus thymidine kinase.Like most other herpesviruses, Epstein-Barr virus (EBV) encodes a virus-induced protein with thymidine kinase (TK) activity. This enzyme catalyzes the transfer of the gamma phosphoryl group of ATP to the 5' hydroxyl of a variety of deoxynucleosides to produce the corresponding nucleoside monophosphate. It is presumed that the further phosphorylations to the diphosphate and triphosphate forms are carried out by cellular kinases. In the case of herpes simplex virus type 1 (HSV-1) and HSV-2, the substrate specificity of the viral TK is much less restricted than that of the mammalian cell TK (12). Thus, in infected cells the viral enzyme initiates the conversion of nucleoside analogs to phosphorylated forms which either are inhibitors of the viral DNA polymerase or are relatively toxic when incorporated into the viral DNA (5). If the EBV TK has a similar broad substrate specificity, it may provide a route to the activation of nucleoside analogs for the production of useful antiviral nucleotides. To examine this possibility, we examined the use of various nucleoside analogs by EBV TK made in a bacterial expression system programed with the cloned EBV DNA sequence encoding this enzyme.Reagents. Nucleoside analogs, thymidine, ATP, morpholinoethanesulfonic acid (MES), and bovine serum albumin (BSA) were purchased from Sigma. [14C] mM spermidine-HCl (pH 7.5) and were lysed by the addition of 300 ,ug of lysozyme per ml. The lysed cells were sonicated briefly to reduce the viscosity before pelleting the cellular debris. The supernatant was loaded directly onto a DEAE Biogel column (4 by 30 cm), and the column was eluted with 1 liter of a linear gradient of salt (0 to 500 mM NaCl). Fractions (6 ml) were collected and assayed for TK activity (see below). Fractions containing TK activity were pooled and applied to a hydroxylapatite column (1 by 8 cm) equilibrated with 1 mM potassium phosphate buffer (pH 6.8). The column was washed in succession with one column volume of the equilibrating buffer, the equilibrating buffer containing 500 mM NaCl, and finally the equilibrating buffer. The column was eluted with a 100-ml linear gradient of 10 to 300 mM potassium phosphate (pH 6.8) (10). Fractions with TK activity were dialyzed against 0.2 mM thymidine-50% glycerol-50 mM Tris (pH 6.8) and were stored at -20°C. The enzyme was judged to be about 30% pure on the basis of the staining intensity of the TK polypeptide on polyacrylamide gels.Standard TK assay. Enzyme activity was determined by the differential binding of phosphorylated versus unphosphorylated nucleoside to positively charged DEAE paper (26). All assay...

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

“…D290E and T298S, which are in site 1, have been suggested to be involved in ATP binding and were tested as controls. Results were similar to those reported by Tung and Summers [27], Mg 2+ is the preferred ion for EBV TK, Mn 2+ is less favoured and Zn 2+ could inhibit its activity, as shown in Figure 3. Among the mutants tested, only D392E revealed an altered pattern of metal-ion usage, where nearly similar activities were found for all three metal ions.…”

“…A biochemical study revealed that EBV TK has K m values of 22 µM for thymidine and 25 µM for ATP [27]. Substrate specificity has also been determined by several groups [27,28]. However, the structurefunction relationship of EBV TK has been addressed rarely.…”

The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase

“…nucleoside analogs. [9][10][11] We thus compared tk from HSV1, varicella-zoster virus (VZV), equine herpes virus type-4 (EHV4) and Epstein-Barr virus (EBV) for their capacity to sensitize tumor cells to ACV, GCV and BVDU.…”

Functional Divergence of Kaposi's Sarcoma-Associated Herpesvirus and Related Gamma-2 Herpesvirus Thymidine Kinases: Novel Cytoplasmic Phosphoproteins That Alter Cellular Morphology and Disrupt Adhesion

“…These highly active enzymes are required for efficient infection of primary epithelial cells (13) and reactivation of lytic replication from neuronal latency (7). In contrast, the gammaherpesvirus TKs are strict thymidine kinases that do not phosphorylate the other nucleosides and have greatly reduced enzymatic activity compared with the alphaherpesvirus TKs (16,20,21,30,46). It is not known whether these enzymes are required for lytic replication in terminally differentiated oral epithelial cells, following lytic reactivation from latently infected B lymphocytes or from endothelial tumor cells in the case of Kaposi's sarcoma-associated herpesvirus (KSHV).…”