[A novel colorimetric test to study the susceptibility of human cytomegalovirus DNA polymerase to foscarnet].
We described a colorimetric method to determine the biochemical phenotype of wild-type and mutated cytomegalovirus (HCMV) DNA polymerases by measuring the incorporation of digoxigenin-labelled nucleotides into the growing DNA chain. Mutations V715M and E756K, which are known to confer foscarnet-resistance, were used as controls. Mutation N495K and a combination of changes K415R and S291P, both observed in foscarnet-resistant isolates, were studied. The mutations were introduced by site-directed mutagenesis into wild-type gene UL54 cloned in an expression vector and then polymerases were synthesised by using a commercially available coupled transcription-translation system. The polymerase activity was measured with and without foscarnet. 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 combination of K415R and S291P, induced a five- and ten-fold decrease in susceptibility to foscarnet, respectively. The results of this non-radioactive assay were consistent with those obtained with the conventional radioactive assay. Therefore, this novel phenotypic method could be useful for the characterisation of mutations that confer HCMV resistance to foscarnet.
Ducancelle
A
A
Service de bactériologie-virologie, hôpital Lariboisière, Assistance publique-hôpitaux de Paris, 2 rue Ambroise-Paré, 75010 Paris, France.
Alain
S
S
Scieux
C
C
Fillet
A-M
AM
Petit
F
F
Sanson-Le Pors
M-J
MJ
Mazeron
M-C
MC
D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000187:drug effects* / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase / Q000187:drug effects / Q000378:metabolism*; D004622:Embryo, Mammalian; D005347:Fibroblasts; D017245:Foscarnet / Q000494:pharmacology*; D006801:Humans; D008826:Microbial Sensitivity Tests
D000998:Antiviral Agents; D017245:Foscarnet; D004259:DNA-Directed DNA Polymerase