The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of α-like DNA polymerases

Lin Huang, Keiko Kumura Ishii, Harmon Zuccola, Amy M. Gehring, Charles B.C. Hwang, James Hogle, Donald M. Coen

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Abstract

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpes-virus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among α-like DNA polymerases. We measured K(m) and k(cat) values for dGTP and ACV triphosphate incorporation and K(i) values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased K(m) values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced k(cat) values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered k(m) and k(cat) values. In most cases, the fold increase in K(i) of ACV triphosphate for dGTP incorporation relative to wild- type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in K(i). The effects of mutations on enzyme kinetics could be explained by using a model of an α-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

Original languageEnglish
Pages (from-to)447-452
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number2
DOIs
Publication statusPublished - 1999 Jan 19

ASJC Scopus subject areas

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