Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional assays.

Twenty amino acid substitutions in the thymidine kinase (TK) of clinical herpes simplex virus type 1 strains were assessed for conferring acyclovir (ACV) resistance. Site-directed mutagenesis, cell-free protein synthesis and protein expression in Escherichia coli were performed to obtain recombinant TK proteins, which were authenticated by Western blotting. A modified enzyme-linked immunosorbent assay (ELISA) was carried out to determine the phosphorylation activity of the mutants towards 5-bromo-2'-deoxyuridine (BrdU). The activity against ACV and deoxythymidine (dT) was analyzed by high performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) following incubation of recombinant TK with ACV and dT. Using ELISA, seven substitutions (G61E, A93V, M121K, R163G, P173del, V238F, G264V) showing negative activity could be classified likely as resistance-related, eleven (Q15K, R20C, R32H, E43A, E43D, R89H, A156V, P269S, G271V, S276N, I326V) with high activity as natural polymorphisms, and two (N244H and N376stop) with low phosphorylation activity. Since the N244H protein did not show any activity towards ACV, but activity towards dT using HPLC/UV, it was classified as TK with altered substrate specificity. In conclusion, the ELISA determining activity towards BrdU is suitable for the characterization of substitutions regarding their significance for resistance. Ambiguous results can be re-assessed by HPLC/UV, which classifies TK with altered substrate specificity.