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Published on: May 4, 2015
Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional
Marisa Kaspar1, Kathrin Bohn-Wippert2, Peter Bellstedt3
1Section Experimental Virology, Institute of Medical Microbiology, German Consulting Laboratory for HSV and VZV, Jena University Clinic, Jena, Germany.
Abstract:
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.
Insights
This study investigated 20 amino acid substitutions in herpes simplex virus type 1 thymidine kinase (TK) for acyclovir resistance. Seven substitutions confer resistance, while others represent natural variations or altered substrate specificity.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is a key target for antiviral drugs like acyclovir (ACV).
- Understanding amino acid substitutions in TK is crucial for predicting and overcoming drug resistance.
Purpose of the Study:
- To assess the impact of 20 amino acid substitutions in HSV-1 TK on acyclovir (ACV) resistance.
- To characterize the phosphorylation activity of mutant TK enzymes towards various substrates.
Main Methods:
- Site-directed mutagenesis and recombinant protein expression in E. coli.
- Enzyme-linked immunosorbent assay (ELISA) for phosphorylation activity against 5-bromo-2'-deoxyuridine (BrdU).
- High-performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) to analyze activity against ACV and deoxythymidine (dT).
Main Results:
- Seven substitutions (G61E, A93V, M121K, R163G, P173del, V238F, G264V) were identified as likely conferring ACV resistance due to negative phosphorylation activity.
- Eleven substitutions (Q15K, R20C, R32H, E43A, E43D, R89H, A156V, P269S, G271V, S276N, I326V) were classified as natural polymorphisms with high activity.
- Two substitutions (N244H, N376stop) showed low phosphorylation activity, with N244H exhibiting altered substrate specificity (active against dT, inactive against ACV).
Conclusions:
- ELISA using BrdU is a reliable method for initial screening of TK substitutions for ACV resistance.
- HPLC/UV is valuable for re-assessing ambiguous results and identifying TK variants with altered substrate specificity, such as N244H.
- This research aids in understanding the molecular basis of acyclovir resistance in HSV-1.
