Substrate Diversity of Herpes Simplex Virus Thymidine Kinase

Abstract

Herpes simplex virus type 1 (HSV 1) 1 thymidine kinase (TK) is a multifunctional enzyme that possesses kinase activities normally performed by three separate cellular enzymes. It phosphorylates thymidine (dT), which is then transformed by cellular kinases to the triphosphorylated DNA building block, and deoxyuridine (dU); both reactions are comparable to the function of human cellular TK. Further, it converts deoxycytidine (dC) to dCMP, as does human deoxycytidine kinase (dCK), and phosphorylates thymidylate (dTMP), as does human TMP kinase (TmpK) (1-3). Moreover, unlike its cellular counterpart human cellular TK, HSV 1 TK is able to phosphorylate pyrimidine, as well as purine analogs, and discloses low stereochemical demands for the ribose moiety, as it also accepts acyclic side chains as phosphoryl group acceptors e.g. (4 -6). These differences in substrate diversity are the crucial molecular basis for the selective treatment of viral infections. Nowadays, the most widely used therapeutic compounds to interfere with a severe HSV 1 infection are the purine analogs acyclovir (ACV) and penciclovir and their prodrugs valaciclovir and famciclovir, respectively. They require HSV 1 TK to be efficiently activated in order to block virus proliferation by inhibition of viral DNA polymerase. HSV 1 TK is the key enzyme in this antiviral strategy. In gene therapy of cancer (7,8) and AIDS (9), HSV 1 TK is used as a suicide enzyme in combination with the purine analog ganciclovir. Another important application is the use of HSV 1 TK as a rescue system in allogeneic bone marrow transplantation-induced graft versus host disease (10). In addition to the significance from a therapeutic point of view, HSV 1 TK seems to be important for the reactivation of the virus from lifelong latent infection in neuronal ganglia (11-13). However, there is evidence that human TK can functionally replace viral TK in terms of reactivation of the virus from latency (14).There are no recognizable sequence similarities between HSV 1 TK and human cellular TK (15). Rather, sequence alignments have detected similarities between herpesvirus TKs and human dCK (16) and to a lesser extent cellular TmpK (17). Despite the limited sequence homology with enzymes of the nucleotide kinase (NK) family, HSV 1 TK shares structural features comprising a parallel five-stranded ␤-sheet and a glycine-rich loop common to all NKs. In the crystal structure, HSV 1 TK is a homodimeric enzyme with 376 amino acids per subunit (18 -20). The two subunits are related by C2 symmetry. The active site is formed by an ATP-and a nucleoside-binding region. The visual representation of the thymidine binding site is depicted in Fig. 1, featuring a complex hydrogen bond network within the active site. The thymine ring makes pairwise hydrogen bond interaction via its 4-carbonyl and 3-NH group with the amide group of the highly conserved Gln-125 and hydrogen bonds with Arg-176 by means of two ordered water molecules. Moreover, the pyrimidine ring of thymidine is fixed between Met-128 and T...

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

“…Thymidinebinding sites are also the regions that are responsible for binding to various nucleoside analogues. Attempts to engineer the thymidine-binding sites to modify the substrate specificities of viral TKs for clinical application have been reported [11]. In this study, the EBV TK nucleoside-binding sites were defined and the biochemical roles of amino acids in these regions were characterized.…”

“…The genes are introduced into tumour cells, which, subsequently, can be killed selectively by the appropriate nucleoside analogues. Much effort has been put into improving the selective killing effect by synthesizing new prodrugs [10] and constructing TK mutants [11].…”

A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing

“…As well as GCV, HSV1 TK also recognizes the nucleoside analogs acyclovir (ACV), penciclovir (PCV), and brivudine (BVDU) as substrates, and accepts the natural nucleosides deoxycytidine (dC), deoxyguanosine (dG), deoxyuridine (dU), deoxythymidine (dT) and thymidylate (dTMP) as substrates [16,17]. Thus, in the tumor cell context, this promiscuity may also influence the GCV cell-killing efficiency due to competition between GCV and the natural substrates of HSV1 TK.…”

2,3‐Dihydroxy‐quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

“…It operates in most eukaryotic cells as well as in several viruses (HSV, HCMV, EBV) [1]. Viral TKs significantly differ in both specificity and selectivity from the human ones and thus represent an attractive target for antiviral therapy [2].Thymidine kinase of Herpes Simplex Virus type 1 (HSV-TK) was successfully targeted by both selective inhibitors and substrate prodrugs which are enzymatically converted to toxic compounds. One of the most common antiviral drug, Acyclovir (ACV), is a HSV-TK substrate which is converted to acyclo-guanosine monophosphate (Acyclo-GMP).…”

“…It operates in most eukaryotic cells as well as in several viruses (HSV, HCMV, EBV) [1]. Viral TKs significantly differ in both specificity and selectivity from the human ones and thus represent an attractive target for antiviral therapy [2].…”