Several drug-resistant strains of herpes simplex virus type 1 (HSV1) isolated in vivo or from tissue culture, have exhibited a mutated thymidine kinase (TK). Moreover, various site-directed-mutagenesis experiments conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hypotheses was generated related to substrate binding of TK at the molecular level. A site-directed-mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine or aciclovir beyond the hydrogen-bonding pattern observed in the crystal structure. While Q125L is only able to phosphorylate thymidine, Q125N accepts thymidine and aciclovir as substrates. Q125E shows no phosphorylation activity. Several mutations identified previously as relevant in drug resistance were studied in an attempt to further understand their role in these processes. Four amino acid positions are described (T63, A168, R176 and C336) that confer drug resistance when mutated ; however, the molecular mechanisms are considerably different in each case. Analysis of the crystal structures and the molecular modeling presented in this paper suggest that T63 is essential for the binding of Mg 2ϩ and thus the catalytic activity of the enzyme, while A168 limits steric accessibility and if mutated to a bulkier residue will exclude binding of larger substrate analogues. R176 appears to be essential for electrostatic balance within the active site, and C336, which is located at the surface of TK and directed toward the ATP-binding site, disrupts the three-dimensional structure of the whole active site by shifting the LID-domain. The present work contributes to a detailed understanding of nucleoside binding to TK, thereby facilitating the rational design of substrates for HSV1 TK and of drug-specific TK for gene therapy.Keywords : herpes simplex virus ; thymidine kinase ; mutation; aciclovir ; resistance.Thymidine kinase (TK) is a key enzyme in the pyrimidine-herpesviral TK isoenzymes is the molecular basis of selective salvage pathway catalyzing the transfer of γ-phosphate from antiviral therapy. Furthermore, HSV1 TK was used recently in ATP to thymidine, to synthesize thymidine monophosphate combination with aciclovir or gangciclovir as suicide enzyme in (dTMP). Herpes viruses encode their own thymidine kinases gene therapy of cancer [2Ϫ4] and AIDS [5]. Therefore, further which differ considerably from the human host cell isoenzyme. understanding of the molecular mechanism of HSV1 TK and its In particular, viral TKs are able to phosphorylate a broad drug-resistant mutants could lead to rational design of antiviral spectrum of pyrimidine and purine substrate analogues including drugs and to customized drug-specific TK for gene therapy. those carrying an acyclic sugar moiety such as the antiviral There are four classes of single mutations of the HSV gedrugs aciclovir and ganciclovir [1]. Compounds such as nome that confer drug resistance. Three of these affect the th...

Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.