DRUG-RESISTANCE OF HERPES-SIMPLEX VIRUS TYPE-1 - STRUCTURAL CONSIDERATIONS AT THE MOLECULAR-LEVEL OF THE THYMIDINE KINASE

Several drug-resistant strains of herpes simplex virus type 1 (HSV1) i solated in viva or from tissue culture, have exhibited a mutated thymi dine kinase (TK). Moreover. various site-directed-mutagenesis experime nts conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hyp otheses was generated related to substrate binding of TK at the molecu lar level. A site-directed-mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine o r aciclovir beyond the hydrogen-bonding pattern observed in the crysta l structure. While Q125L is only able to phosphorylate thymidine, Q125 N accepts thymidine and aciclovir as substrates. Q125E shows no phosph orylation activity. Several mutations identified previously as relevan t 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 c ase. Analysis of the crystal structures and the molecular modeling pre sented in this paper suggest that T63 is essential for the binding of Mg2+ 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 fo r electrostatic balance within the active site, and C336, which is loc ated at the surface of TK and directed toward the ATP-binding site, di srupts the three-dimensional structure of the whole active site by shi fting the LTD-domain. The present work contributes to a detailed under standing of nucleoside binding to TK, thereby facilitating the rationa l design of substrates for HSV1 TK and of drug-specific TK for gene th erapy.