Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside 5'-triphosphates.
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.
Ueno
T
T
Experimental Retrovirology Section, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Shirasaka
T
T
Mitsuya
H
H
D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D054306:Dideoxynucleotides; D018432:Drug Resistance, Multiple / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
D000998:Antiviral Agents; D017931:DNA Primers; D004279:DNA, Viral; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C037773:2',3'-dideoxyadenosine triphosphate; D015215:Zidovudine; C031709:2',3'-dideoxyguanosine 5'-triphosphate; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate