Genetic analysis of a ganciclovir-resistant human cytomegalovirus mutant

We isolated a ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) from a laboratory strain, AD169, and analysed the mutant. Attempts were also ma de to identify directly the mutated gene. The 50% inhibitory concentration (IC50) of GCV for the mutant strain was five times higher than that of the wild-type strain. The mutant strain showed similar sensitivity to phosphono acetic acid and cidofovir as the wild-type strain. These data suggest mutat ion in the UL97 gene encoding for the phosphotransferase that phosphorylate s GCV. Molecular analysis of the mutant strain revealed that a single base substitution of adenine by cytosine occurred at the 1796 nucleotide positio n of the UL97 gene region, resulting in the substitution of lysine by threo nine at codon 599 in the UL97 gene product. Marker transfer experiment conf irmed that this mutation conferred HCMV resistance to GCV. The mutation at codon 599 was easily identified by means of RsaI digestion of the selected PCR product. (C) 1998 Published by Elsevier Science B.V. All rights reserve d.