Phenotypic characterisation of cytomegalovirus DNA polymerase: a method to study cytomegalovirus isolates resistant to foscarnet

• Published in: Journal of virological methods 2005-05, Vol.125 (2), p.145-151
• Main Authors: Ducancelle, Alexandra, Gravisse, Jérôme, Alain, Sophie, Fillet, Anne-Marie, Petit, Françoise, Pors, Marie-José Sanson Le, Mazeron, Marie-Christine
• Format: Article
• Language: English
• Subjects: Bacteriology; Biological and medical sciences; Cytomegalovirus; Cytomegalovirus - genetics; Cytomegalovirus - isolation & purification; Cytomegalovirus Infections; Cytomegalovirus Infections - virology; DNA polymerase; DNA-Directed DNA Polymerase; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - isolation & purification; Drug Resistance, Viral; Drug Resistance, Viral - genetics; Foscarnet; Foscarnet - pharmacology; Fundamental and applied biological sciences. Psychology; Genetic Variation; Human cytomegalovirus; Human health and pathology; Infectious diseases; Life Sciences; Microbiology; Microbiology and Parasitology; Resistance; Techniques used in virology; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.01.005

A phenotypic method was developed to test mutations in the human cytomegalovirus (HCMV) DNA polymerase gene ( UL54) suspected to confer resistance to foscarnet. This method was used to determine the biochemical phenotype of wild-type and mutated HCMV DNA polymerases that had been synthesised in vitro as follows. The UL54 genes were amplified from foscarnet-resistant and -sensitive isolates by PCR and the products were cloned into an expression vector under the control of a T7 promoter. Mutations were introduced by site-directed mutagenesis into wild-type gene UL54 and then polymerases were synthesised by using a commercially available coupled transcription/translation system. Polymerase activity was measured with and without foscarnet by detecting the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. It was found that the activity of polymerases containing the V715M or E756K mutations was inhibited by foscarnet at concentrations 70- and 30-fold higher than that of wild-type polymerase, respectively. Change N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, induced a 5- and 10-fold decrease in susceptibility to foscarnet, respectively. This non-radioactive phenotypic assay could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.