Significance of amino acid substitutions in the thymidine kinase gene of herpes simplex virus type 1 for resistance.
ABSTRACT 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.
- SourceAvailable from: Etienne Muller[show abstract] [hide abstract]
ABSTRACT: Herpes simplex virus type 2 (HSV-2) is currently the leading cause of Genital Ulcer Disease (GUD) both globally and within South Africa. HSV-2 infections are most often treated with acyclovir (ACV), a guanosine nucleoside analogue that requires phosphorylation by virus-encoded thymidine kinase (TK). Resistance to ACV is mainly due to mutations in the viral UL23 gene that codes for TK. ACV was added as part of the first-line syndromic management treatment algorithm for GUD in South Africa in late 2008. In order to assess the prevalence of TK-associated ACV resistance among HSV-2 virions detected in genital ulcer specimens, pre- and post-introduction of ACV, we amplified and fully sequenced the UL23 gene of 254 HSV-2 positive specimens obtained from participants in GUD aetiological surveys conducted between 2007 and 2011 in Johannesburg, South Africa. We identified 63 nucleotide mutations in the UL23 genes analysed, that resulted in 30 silent mutations and 32 amino acid changes. A large proportion (41%) of these amino acid changes were due to previously described natural polymorphisms that occur in both sensitive and resistant HSV strains. In addition, we identified 19 unknown amino acid changes in 30 samples that have not been described before. All mutations detected were outside the recognised TK conserved domains where ACV resistance mutations typically occur. No frameshift mutations or mutations causing stop codons were identified in those UL23 genes analysed. Importantly, no evidence was found of known ACV resistance mutations in HSV-2 following the addition of ACV as first-line therapy for GUD.Journal of Antivirals and Antiretrovirals 07/2013; 5(4):080-084.