A POINT MUTATION IN THE HUMAN CYTOMEGALOVIRUS DNA-POLYMERASE GENE SELECTED IN-VITRO BY CIDOFOVIR CONFERS A SLOW REPLICATION PHENOTYPE IN CELL-CULTURE

In cell culture, cidofovir (CDV) was used to select a human cytomegalo virus (HCMV) strain with decreased drug susceptibility The genotypic c haracterization of this virus revealed a single base substitution resu lting in a K513N amino acid alteration in the viral DNA polymerase (UL 54). Performed in parallel, the selection of HCMV for replication in t he presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substituti on in DNA polymerase. Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains. To precisel y elucidate their role in drug resistance, corresponding recombinant m utant viruses were generated by recombination of nine overlapping vira l DNA fragments. The K513N recombinant virus showed 13- and 6.5-fold d ecreased susceptibility to CDV and GCV in vitro, respectively, compare d with the wild-type recombinant virus. Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, fos carnet, and adefovir. A multiplicative interaction of the K513N and V8 12L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant Virus expressing both mutations. in Vi tro replication kinetic experiments revealed that the K513N mutation s ignificantly decreased HCMV replication capacity Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific ac tivity in comparison with the wild-type enzyme and was severely impair ed in its 3'-5' exonuclease function. Unexpectedly, the K513N mutant e nzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-t riphosphate, However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient tran sfection/infection assay, suggesting that the DNA replication of the K 513N mutant virus is less sensitive to the corresponding inhibitors. ( C) 1998 Academic Press.