Identification of resistance-associated HCMV UL97- and UL54-mutations and a UL97-polymporphism with impact on phenotypic drug-resistance

•Patients after stem cell transplantation developing drug-resistant HCMV infections have a high risk for severe disease.•To define weather mutations are associated to drug-resistance, they have to be confirmed by marker transfer analysis.•We describe the generation and characterisation of mutants containing newly found UL97 and UL54 mutations.•In addition, combinations of UL97 and UL54 mutations were characterised to investigate the influence on drug resistance.•This information may lead to a better understanding and classification of newly emerging UL97-and UL54 mutations.

BackgroundHuman cytomegalovirus (HCMV) drug-resistance remains of high clinical importance. While UL97-mutations can confer ganciclovir-resistance, UL54-mutations can be associated with resistance to ganciclovir, foscarnet and/or cidofovir.ObjectiveThree UL97-mutations (A619V, P468Q, del597-599), three UL54-mutations (V715A, A492D, L516W) and two UL97/UL54-mutation combinations (A594TUL97+V715MUL54; A591VUL97+D515EUL54, L516MUL54, I521TUL54) were characterised phenotypically. All mutations were introduced into the bacterial artificial chromosome (BAC) TB40-BACKL7-UL32EGFP. A revertant of HCMV-TB40-BACKL7-UL32EGFP/A591VUL97+D515EUL54, L516MUL54, I521TUL54 was generated.ResultsThe UL97-mutation del597-599 showed GCV-resistance while A619V and P468Q were drug-sensitive. The UL54-mutation V715A was FOS-resistant/CDV-hypersensitive and L516W was GCV/CDV cross-resistant. Mutation A594TUL97+V715MUL54 showed GCV/FOS cross-resistance. HCMV-BACKL7-UL32EGFP/A591VUL97+D515EUL54,L516MUL54, I521TUL54 was GCV/CDV cross-resistant with a remarkably increased GCV-ratio compared to a strain where only the UL54-mutations D515E+L516M+I521T were present. Since the revertant was drug-sensitive again, the increased drug-ratio is supposed to be due to the UL97-polymorphism A591V.ConclusionPhenotypic characterisation of newly detected mutations in UL97 and UL54 remain of high importance. Only mutations with a confirmed phenotype allow reliable interpretation of genotypic methods. Here, we provide the first description of a UL97-polymorphism that contributes to the overall drug-resistance when combined with resistance-associated UL54-mutations. The finding shows the high importance to look at mutations in the context of their genetic background.