ABSTRACT
Recombinant phenotyping of cytomegalovirus (CMV) pol region III mutations from clinical specimens showed that T813S and G841A each conferred foscarnet resistance and approximately threefold increased ganciclovir resistance; adding the UL97 mutation C592G increased ganciclovir resistance to approximately sixfold. Bacterial artificial chromosome CMV clones containing pol mutation L845P were nonviable unless repaired with the wild-type sequence.
Subject(s)
Antiviral Agents/pharmacology , Cytomegalovirus/drug effects , DNA-Directed DNA Polymerase/genetics , Drug Resistance, Viral/genetics , Mutation , Cytomegalovirus/genetics , Cytomegalovirus/growth & development , Cytomegalovirus Infections/virology , Foscarnet/pharmacology , Ganciclovir/pharmacology , Genotype , Humans , PhenotypeABSTRACT
The cytomegalovirus UL97 kinase inhibitor maribavir suppressed viral growth more effectively in lung fibroblasts than in skin fibroblasts, and some cellular kinase inhibitors enhanced its antiviral activity. These effects influence the phenotypic assay of drug susceptibility and suggest the possibility of therapeutically useful combinations of maribavir and cellular kinase inhibitors.
Subject(s)
Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Cytomegalovirus/drug effects , Cytomegalovirus/growth & development , Ribonucleosides/pharmacology , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Cells, Cultured , Cytomegalovirus/enzymology , Fibroblasts/virology , Genes, Reporter , Humans , Lung/cytology , Lung/virology , Skin/cytology , Skin/virology , Virus Cultivation/methodsABSTRACT
A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.
