title: Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors.
abstract: A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
authors.affiliation: Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Israel.
authors.forename: S
authors.initials: S
authors.lastname: Loya
authors.forename: M
authors.initials: M
authors.lastname: Bakhanashvili
authors.forename: R
authors.initials: R
authors.lastname: Tal
authors.forename: S H
authors.initials: SH
authors.lastname: Hughes
authors.forename: P L
authors.initials: PL
authors.lastname: Boyer
authors.forename: A
authors.initials: A
authors.lastname: Hizi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004279:DNA, Viral / Q000096:biosynthesis; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D019829:Nevirapine; D010618:Phenanthrolines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000378:metabolism; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000494:pharmacology; D014443:Tyrosine / Q000502:physiology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D010618:Phenanthrolines; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; D014443:Tyrosine; D015215:Zidovudine; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; C017216:2',3'-dideoxythymidine triphosphate; C025205:1,10-phenanthroline