A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives.
Ganciclovir-resistant mutant 759rD100 derived from human cytomegalovirus strain AD169 contains two resistance mutations, one of which is in the UL97 gene and results in decreased ganciclovir phosphorylation in infected cells [V. Sullivan, C. L. Talarico, S. C. Stanat, M. Davis, D. M. Coen, and K. K. Biron, Nature (London) 358:162-164, 1992]. In the present study, we mapped the second mutation to a 4.1-kb DNA fragment containing the DNA polymerase gene and showed that it confers ganciclovir resistance without impairing phosphorylation. Sequence analysis of the 4.1-kb region revealed a single nucleotide change that resulted in a glycine-to-alanine substitution at position 987 within conserved region V of the DNA polymerase. Recombinant viruses constructed to contain the DNA polymerase mutation but not the phosphorylation defect displayed intermediate resistance (4- to 6-fold) to ganciclovir relative to the original mutant 759rD100 (22-fold); the recombinant viruses also displayed resistance to ganciclovir cyclic phosphate (7-fold), 1-(dihydroxy-2-propoxymethyl)-cytosine (12-fold), and the phosphonylmethoxyalkyl derivatives (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (8- to 10-fold). However, the recombinant viruses remained susceptible to certain related compounds. These results imply that the human cytomegalovirus DNA polymerase is a selective target for the antiviral activities of ganciclovir, certain of its derivatives and phosphonomethoxyalkyl derivatives; support a role for region V in substrate recognition; and suggest the possibility of clinical resistance of human cytomegalovirus to these compounds because of polymerase mutations.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
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Biron
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003587:Cytomegalovirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics*; D015774:Ganciclovir / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005814:Genes, Viral; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010957:Plasmids; D017354:Point Mutation; D011995:Recombination, Genetic; D010948:Viral Plaque Assay
D005819:Genetic Markers; D009943:Organophosphorus Compounds; D004259:DNA-Directed DNA Polymerase; D015774:Ganciclovir