Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine.
A molecular clone containing the wild-type reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) was constructed, and site-directed mutagenesis was used to introduce mutations--Leu74-->Val (L74V), T215Y, and the combination L74V/T215Y--into the RT coding region. The proteins were purified by immunoaffinity chromatography. Assays were performed with mutant and wild-type RT to determine substrate and inhibitor specificity. All three mutant enzymes catalyzed the incorporation of substrate 2'-deoxynucleoside 5'-triphosphates (dNTPs) as efficiently as wild-type HIV-1 RT. Small changes were observed in the Km values for dNTPs with all three mutant enzymes, while more significant changes were noted in sensitivity to nucleoside 5'-triphosphate analogues that inhibit the enzyme activity. Results suggest that altered substrate recognition by the HIV-1 RT is involved in the mechanism of resistance.
Martin
J L
JL
Division of Virology, Burroughs Wellcome Company, Research Triangle Park, NC 27709.
Wilson
J E
JE
Haynes
R L
RL
Furman
P A
PA
D001483:Base Sequence; D003001:Cloning, Molecular; D003854:Deoxyribonucleotides / Q000378:metabolism; D016049:Didanosine / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors
D003854:Deoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016049:Didanosine