title: Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization.
abstract: "BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio.
authors.forename: P S
authors.initials: PS
authors.lastname: Jacques
authors.forename: B M
authors.initials: BM
authors.lastname: Wöhrl
authors.forename: M
authors.initials: M
authors.lastname: Ottmann
authors.forename: J L
authors.initials: JL
authors.lastname: Darlix
authors.forename: S F
authors.initials: SF
authors.lastname: Le Grice
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000502:physiology; D013329:Structure-Activity Relationship; D014364:Tryptophan
chemical_list: D014364:Tryptophan; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H