title: The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases.
abstract: Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus 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 alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki 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 Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: L
authors.initials: L
authors.lastname: Huang
authors.forename: K K
authors.initials: KK
authors.lastname: Ishii
authors.forename: H
authors.initials: H
authors.lastname: Zuccola
authors.forename: A M
authors.initials: AM
authors.lastname: Gehring
authors.forename: C B
authors.initials: CB
authors.lastname: Hwang
authors.forename: J
authors.initials: J
authors.lastname: Hogle
authors.forename: D M
authors.initials: DM
authors.lastname: Coen
mesh_terms: D000212:Acyclovir / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics*; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006566:Herpesviridae Infections / Q000188:drug therapy; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000998:Antiviral Agents; D003848:Deoxyguanine Nucleotides; D004791:Enzyme Inhibitors; C029603:deoxyguanosine triphosphate; D004256:DNA Polymerase I; D000212:Acyclovir