HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors.
The parameters governing the polymerization mechanism of reverse transcriptase containing the tyrosine to cysteine mutation at position 181 (Y181C) were determined using pre-steady-state techniques. The pathway for single nucleotide incorporation catalyzed by Y181C is similar to that determined for wild-type RT where a rate-limiting conformational change precedes fast chemistry and is followed by slow steady-state release of the primer/template. The Y181C mutant enzyme binds a 25/45-mer duplex DNA tightly with a Kd of 11 nM. However, the Y181C mutation weakens the nucleotide affinity 2-3-fold relative to the wild-type complex. We also determined the parameters governing the mechanism of nonnucleoside inhibitor resistance with Y181C. The Kd value of Nevirapine with the mutant E.DNA complex increased approximately 500-fold. The decreased affinity of Nevirapine for the mutant enzyme is a consequence of a faster inhibitor dissociation rate from the enzyme complex of Y181C relative to that of the wild-type. The E.DNA complex of Y181C may be saturated with Nevirapine, and the I.E.DNA complex is capable of a maximum incorporation rate of 0.1 s-1 (a 10-fold faster rate than that of the wild-type I.E.DNA complex). The overall two-step binding of nucleotide to Y181C in the presence of Nevirapine remains unaffected.
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
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Spence
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Anderson
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Johnson
D000998:Antiviral Agents / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004351:Drug Resistance; D054303:HIV Reverse Transcriptase; D019829:Nevirapine; D011725:Pyridines / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism*
D000998:Antiviral Agents; D003838:Deoxyadenine Nucleotides; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D004247:DNA; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate