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 cytomegalovirus (HCMV) strain with decreased drug susceptibility. The genotypic characterization of this virus revealed a single base substitution resulting in a K513N amino acid alteration in the viral DNA polymerase (UL54). Performed in parallel, the selection of HCMV for replication in the presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substitution in DNA polymerase. Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains. To precisely elucidate their role in drug resistance, corresponding recombinant mutant viruses were generated by recombination of nine overlapping viral DNA fragments. The K513N recombinant virus showed 13- and 6.5-fold decreased susceptibility to CDV and GCV in vitro, respectively, compared with the wild-type recombinant virus. Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, foscarnet, and adefovir. A multiplicative interaction of the K513N and V812L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant virus expressing both mutations. In vitro replication kinetic experiments revealed that the K513N mutation significantly decreased HCMV replication capacity. Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific activity in comparison with the wild-type enzyme and was severely impaired in its 3'-5' exonuclease function. Unexpectedly, the K513N mutant enzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-triphosphate. However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient transfection/infection assay, suggesting that the DNA replication of the K513N mutant virus is less sensitive to the corresponding inhibitors.
Gilead Sciences, 333 Lakeside Drive, Foster City, California, 94404, USA. tomas_cihlar@gilead.com
T
T
Cihlar
M D
MD
Fuller
A S
AS
Mulato
J M
JM
Cherrington
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D000077404:Cidofovir; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D003596:Cytosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000096:biosynthesis / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000502:physiology; D004352:Drug Resistance, Microbial / Q000235:genetics; D004794:Enzyme Repression; D005092:Exonucleases / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology; D008969:Molecular Sequence Data; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010641:Phenotype; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins; D014779:Virus Replication / Q000235:genetics
D000998:Antiviral Agents; D004279:DNA, Viral; D063065:Organophosphonates; D009943:Organophosphorus Compounds; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D003596:Cytosine; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; C022650:spleen exonuclease; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D000077404:Cidofovir; D015774:Ganciclovir