       Document 0454
 DOCN  M9610454
 TI    Enzymatic characterization of human immunodeficiency virus type 1
       reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside
       5'-triphosphates.
 DT    9601
 AU    Ueno T; Shirasaka T; Mitsuya H; Experimental Retrovirology Section, NCI,
       National Institutes of; Health, Bethesda, Maryland 20892, USA.
 SO    J Biol Chem. 1995 Oct 6;270(40):23605-11. Unique Identifier : AIDSLINE
       MED/96007506
 AB    A set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the
       polymerase domain of reverse transcriptase (RT) of human
       immunodeficiency virus type 1 (HIV-1), which confers on the virus a
       reduced sensitivity to multiple therapeutic dideoxynucleosides (ddNs),
       has been identified. In this study, we defined the biochemical
       properties of RT with such mutations by using site-directed mutagenesis,
       overproduction of recombinant RTs, and steady-state kinetic analyses. A
       single mutation, Q151M, which developed first among the five mutations
       in patients receiving therapy, most profoundly reduced the sensitivity
       of RT to multiple ddN 5'-triphosphate (ddNTPs). Addition of other
       mutations to Q151M further reduced the sensitivity of RT to ddNTPs. RT
       with the five mutations proved to be resistant by 65-fold to
       3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP), 12-fold to
       ddCTP, 8.8-fold to ddATP, and 3.3-fold to 2',3'-dideoxyguanosine
       5'-triphosphate (ddGTP), compared with wild-type RT (RTwt). Steady-state
       kinetic studies revealed comparable catalytic efficiency (kcat/Km) of
       RTs carrying combined mutations as compared with that of RTwt (<
       3-fold), although a marked difference was noted in inhibition constants
       (Ki) (e.g. Ki of a mutant RT carrying the five mutations was 62-fold
       higher for AZTTP than that of RTwt). Thus, we conclude that the
       alteration of RT's substrate recognition, caused by these mutations,
       accounts for the observed multi-ddN resistance of HIV-1. The features of
       multi-ddNTP-resistant RTs should provide insights into the molecular
       mechanism of RT discriminating ddNTPs from natural substrates.
 DE    Antiviral Agents/PHARMACOLOGY  Base Sequence  Binding Sites/GENETICS
       Deoxyadenine Nucleotides/PHARMACOLOGY  Deoxyguanine
       Nucleotides/PHARMACOLOGY  Deoxyribonucleotides/*PHARMACOLOGY  Drug
       Resistance, Multiple/GENETICS  DNA Primers/GENETICS  DNA, Viral/GENETICS
       Human  HIV Infections/DRUG THERAPY/VIROLOGY  HIV-1/DRUG
       EFFECTS/*ENZYMOLOGY/GENETICS  In Vitro  Kinetics  Molecular Sequence
       Data  Mutagenesis, Site-Directed  Point Mutation  Reverse Transcriptase
       Inhibitors/*PHARMACOLOGY  RNA-Directed DNA
       Polymerase/*GENETICS/*METABOLISM  Thymine Nucleotides/PHARMACOLOGY
       Zidovudine/ANALOGS & DERIVATIVES/PHARMACOLOGY  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

