title: Fidelity analysis of HIV-1 reverse transcriptase mutants with an altered amino-acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184.
abstract: Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV-1. The effects of this kind of substitution were determined in a lacZ-based assay using HIV-1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184-->Val and Glu89-->Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC-resistant Met184-->Val RT mutant an almost wild-type level of overall mutation frequency was observed, while the foscarnet-resistant RTs harbouring the Glu89-->Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183-->Phe mutant RT displayed a slightly lower fidelity than wild-type RT. Conversely, the ddI-resistant RT mutant containing the Leu74-->Val mutation showed a 3.5-fold higher fidelity compared to the wild-type enzyme. Finally, the Tyr115-->Ala substitution rendered the enzyme substantially more error-prone for DNA polymerization. These results correlate with three-dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV-1 RT.
authors.affiliation: Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
authors.forename: H
authors.initials: H
authors.lastname: Jonckheere
authors.forename: E
authors.initials: E
authors.lastname: De Clercq
authors.forename: J
authors.initials: J
authors.lastname: Anné
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001704:Biopolymers; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012689:Sequence Homology, Nucleic Acid
chemical_list: D000596:Amino Acids; D001704:Biopolymers; D054303:HIV Reverse Transcriptase