Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains.
Various DNA changes located within a restricted region of the UL97 open reading frame were shown to be associated with the resistance of cytomegalovirus strains to ganciclovir (GCV). In order to analyse this UL97 region in sensitive and GCV-resistant strains, a non-radioactive sequencing assay (Promega, Madison, WI, USA) which combines the dideoxy visualisation by silver-staining of the gel was used. Using this assay, polymerase chain reaction products from results were obtained within 1 day. Point mutations modifying the amino acid sequence of the putative UL97 catalytic site were detected in three isolates. These led to an alanine to valine substitution in residue 594 in one strain with reduced GCV sensitivity, and to a cysteine to glycine substitution in residue 592 in two GCV-resistant isolates. These mutations were different from the DNA changes previously mapped in GCV-resistant laboratory or field strains. No amino acid substitution in the UL97 catalytic site was found in GCV-sensitive isolates. Transfer marker experiments are in progress in order to test the significance of these DNA changes for GCV resistance. This rapid non-radioactive sequencing protocol could be a useful tool for analysing the UL97 region encoding the putative UL97 catalytic site of clinical isolates.
Alain
S
S
Service de Bactériologie-Virologie, Hôpital Lariboisière, Université Paris VII, France.
Mazeron
M C
MC
Pépin
J M
JM
Bergmann
J F
JF
Narwa
R
R
Raskine
L
L
Sanson-Le Pors
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics*; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D017354:Point Mutation / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D004279:DNA, Viral; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir