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

Abstract

Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

Antiherpesvirus Activities of Two Novel 4′-Thiothymidine Derivatives, KAY-2-41 and KAH-39-149, Are Dependent on Viral and Cellular Thymidine Kinases

“…However, no replacement of the proline residue was tolerated at position 173 of HSV-1 TK (corresponding to Pro174 in HSV-2 and Pro326 in HVS), whereas the tyrosine residue at position 172 (HSV-1) could be replaced by the structurally related phenylalanine only (42). Consistent with this study, Wu et al (43) reported that in EBV, the full activity of the TK was retained only when a tyrosine residue replaced the phenylalanine at this position. Thus, the very limited substitution of proline and tyrosine/phenylalanine in the conserved site IV (i.e., nucleosidebinding site) suggests that these residues are essential for maintaining functional TK activity (42,43).…”

Activity and Mechanism of Action of HDVD, a Novel Pyrimidine Nucleoside Derivative with High Levels of Selectivity and Potency against Gammaherpesviruses

“…Comparative TK sequence analysis of 12 herpesviruses, including HSV-1, EBV, and HVS, allowed the identification of five conserved sites in this enzyme, i.e., the putative ATP-binding pocket (site I), the active site (site II), the nucleoside-binding domains (sites III and IV), and the arginine-rich domain which is believed to bind substrate phosphoryl groups (site V) (22,41). The majority of the mutations in the HDVD r HSV-1, HVS, and MHV-68 mutants were mapped in nonconserved regions of the TK, with the exception of one MHV-68 and one HVS mutant, which bore a mutation in the conserved site II (E358D) and site IV (P326T), respectively (Fig.…”

“…This region contains the amino acid motif Val-Phe-Pro, which is highly conserved in gammaherpesviruses, and the proline is conserved in all herpesviruses. Also, the crucial role of the phenylalanine in this motif for maintaining EBV TK activity due to an essential interaction with the pyrimidine nucleoside substrate has been demonstrated previously (22,47). The P326T mutation maps into the Val-Phe-Pro motif.…”

“…Therefore, thymidine analogs may be the most suitable antiviral agents to be activated by the EBV-encoded TK enzyme (18,19). Similar to EBV TK, TKs of other gammaherpesviruses, such as KSHV and herpesvirus saimiri (HVS), have been found to possess comparable substrate specificities (17,(20)(21)(22)(23). In addition, a closely related murine gammaherpesvirus (murine herpesvirus 68 [MHV-68]), encoding a TK with significant homology to the TKs of both EBV and KSHV, is used in a mouse model of infection for the evaluation of antiviral drugs against gammaherpesviruses (24)(25)(26).…”

Spectrum of Activity and Mechanisms of Resistance of Various Nucleoside Derivatives against Gammaherpesviruses

“…Yet structure-function studies concerning the EBV and KSHV TKs have been described. This has been achieved by the engineering of different viral TK mutants by site-directed mutagenesis in order to characterize essential residues in the conserved ATP and substrate binding site of the EBV TK (32,33) and investigate the contributions of the N-and C-terminal regions of KSHV TK (34).…”