Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance

The analysis of the viral thymidine kinase (TK) genotype is of rising significance for testing resistance of herpes simplex virus (HSV) to antivirals especially acyclovir. However, numerous of the described amino acid (aa) substitutions are diagnostically less conclusive because of the pronounced natural polymorphism of this gene. In this study, several aa substitutions in the TK sequence of HSV-1 with unclear significance for resistance were analyzed by expression of recombinant TK proteins and determination of enzymatic activity on the basis of an enzyme linked immunosorbent assay using bromodeoxyuridine (BrdU) as TK substrate. The recombinant TK wild-type protein resulted in high TK activity and TK mutant with stop of translation showed negative results. The recombinant TK proteins containing the aa substitutions R41H or V348I had high phosphorylation activities suggesting most likely natural gene polymorphisms. By contrast, the aa changes Y53H, L139V, R163H, L298A and L315S were accompanied by negative or weakly positive TK activities indicating resistance association. In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains. (C) 2012 Elsevier B.V. All rights reserved.