title: The K65R mutant reverse transcriptase of HIV-1 cross-resistant to 2', 3'-dideoxycytidine, 2',3'-dideoxy-3'-thiacytidine, and 2',3'-dideoxyinosine shows reduced sensitivity to specific dideoxynucleoside triphosphate inhibitors in vitro.
abstract: The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) encodes cross-resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'-thiacytidine (3TC), and 2',3'-dideoxyinosine (ddI). We characterized the in vitro sensitivities of recombinant wild type (wt) and K65R mutant RT to dideoxynucleoside triphosphate (ddNTP) inhibitors, using a variety of primer-templates. With poly(rA)-oligo(dT), the K65R mutant showed slight increases in Ki for ddTTP and 3'-azido, 3'-deoxythymidine triphosphate (AZTTP) compared to wt RT, but neither wt nor K65R RT was inhibited by ddCTP or ddATP. With poly(rI)-oligo(dC), the K65R mutant showed a 2-fold increase in Km for dCTP and a 20-fold increase in Ki for ddCTP compared to wt, whereas ddATP, ddTTP, and AZTTP failed to inhibit either enzyme. With a heteropolymeric primer-template, the K65R mutant showed 10-fold reduced sensitivities to ddCTP, 3TCTP, and ddATP, and 4-fold reduced sensitivity to AZTTP, compared to wt. In contrast, both enzymes were equally inhibited by ddTTP and ddGTP. HIV-1 cross-resistance to ddC/3TC/ddI resulting from the K65R mutation may therefore involve selective alterations in substrate/inhibitor recognition. Additionally, competitive inhibition by ddNTPs noncomplementary to the template base appears to be unimportant in the mechanism of inhibition of HIV-1 RT by dideoxynucleoside analogs.
authors.affiliation: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Gu
authors.forename: R S
authors.initials: RS
authors.lastname: Fletcher
authors.forename: E J
authors.initials: EJ
authors.lastname: Arts
authors.forename: M A
authors.initials: MA
authors.lastname: Wainberg
authors.forename: M A
authors.initials: MA
authors.lastname: Parniak
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D016049:Didanosine / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D019259:Lamivudine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011119:Polynucleotides; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity; D013698:Templates, Genetic; D016047:Zalcitabine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D017931:DNA Primers; D011119:Polynucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; D016047:Zalcitabine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016049:Didanosine