Artikel
Novel procedure to assess resistance relevance of mutations in the Thymidine Kinase Gene of Herpes Simplex Virus Type 1
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Autoren
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Marisa Kaspar
- Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Jena
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Kathrin Bohn-Wippert
- Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Jena
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Peter Bellstedt
- Leibniz Institute on Aging – Fritz Lipman Institute e. V., Jena
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Matthias Görlach
- Leibniz Institute on Aging – Fritz Lipman Institute e. V., Jena
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Andreas Sauerbrei
- Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Jena
| Veröffentlicht: | 30. September 2016 |
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Gliederung
Text
Introduction: In immunocompromised patients, the prevalence of acyclovir (ACV) resistant herpes simplex virus type 1 (HSV-1) reaches amounts up to nearly 50%. For successful antiviral therapy, the rapid availability of diagnostic genotypic resistance data is of paramount importance. To meet this requirement, a broad knowledge of natural polymorphisms and resistance conferring mutations particularly in the thymidine kinase (TK) gene is crucial.
Objectives: The aim of this study was to assess 20 amino acid (aa) substitutions in the TK gene of clinically characterized HSV-1 strains with respect to their significance for conferring ACV resistance. Following preparation of individual recombinant TK protein mutants, the enzymatic activity of the TK mutants was evaluated.
Materials and Methods: Site-directed mutagenesis, cell-free protein synthesis and protein expression in E. coli were performed to obtain recombinant TK proteins. Authentication of expressed protein was achieved by western blot analysis. A modified DiviTumTM assay was carried out to determine the phosphorylation activity of the individual TK mutants towards 5-bromo-2’-deoxyuridine (BrdU). In order to evaluate the activity against ACV, the amount of ACV, ACV monophosphate and adenosine tri-/diphosphate was determined by high performance liquid chromatography/ultraviolet spectroscopy (HPLC/UV) following incubation of recombinant TK with ACV.
Results: Using the DiviTumTM assay, eleven aa changes in the TK gene were characterized as natural polymorphisms, six as resistance-associated mutations and three as polymorphisms with low phosphorylation activity. Additionally, the HPLC/UV method was established as a novel functional TK assay to validate the significance of aa substitutions with respect to resistance.
Conclusion: The modified DiviTumTM TK functional assay is suitable for the characterization of resistance-conferring aa substitutions in the HSV-1 TK. As this test does not use ACV as substrate, ambiguous results can be re-assessed by the implementation of the assay involving HPLC/UV, which, however, is more difficult to perform. More importantly, the application of purified recombinant proteins and the usage of ACV as substrate in this assay render this method highly reliable.
