GCV resistance due to the mutation A594P in the cytomegalovirus protein UL97 is partially reconstituted by a second mutation at D605E.

A ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) was isolated from an AIDS patient. Molecular analysis of the HCMV UL97 gene revealed two point mutations, A594P and D605E, respectively. In order to evaluate quantitatively the impact of the individual mutations on GCV phosphorylation, recombinant vaccinia viruses (rVVs) were generated carrying either the two mutations (rVV-594/605) or only one mutation (rVV-594 or rVV-605, respectively). In cells infected with the rVV-594/605 double mutant, the GCV phosphorylation was decreased to 50% compared with the phosphorylation in cells infected with the rVV-UL97 wild-type. In cells infected with the rVV-594, however, the GCV phosphorylation was further decreased to 30%. Interestingly, the mutation D605E led to an even better GCV phosphorylation than that measured in cells infected with the rVV-UL97 wild type. These results were confirmed by plaque reduction assays, indicating that rVV-594 was more resistant to GCV than rVV-594/605. In contrast, rVV-605 was more sensitive to GCV than the rVV-UL97 wild type. Therefore, our results demonstrated for the first time that compensatory mutations can also occur in HCMV, as already shown for human immunodeficiency virus type 1.