Identification of the amino acid in the human immunodeficiency virus type 1 reverse transcriptase involved in the pyrophosphate binding of antiviral nucleoside triphosphate analogs and phosphonoformate. Implications for multiple drug resistance.
A recombinant clone of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with reduced sensitivity to 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) and phosphonoformate (PFA), a pyrophosphate analog, has been obtained from the RNA of HTLV-IIIB infected cells using the polymerase chain reaction. The mutant HIV-1 RT retained polymerase activity and was cross-resistant to triphosphate forms of other nucleoside analogs including 2',3'-dideoxycytidine 5'-triphosphate, 2',3'-dideoxyadenosine 5'-triphosphate, and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (D4TTP), but remained sensitive to the non-nucleoside HIV-1 RT inhibitors, such as nevirapine and TIBO R82150. Sequence analysis of the mutant HIV-1 RT revealed a single amino acid substitution (Val-->Ala) at amino acid 90. The substitution of amino acid 90 by the closely related amino acids, such as Thr and Gly, also showed decreased sensitivity to AZTTP, D4TTP, and PFA. All these mutations at amino acid 90 also caused an alteration of Km for thymidine triphosphate. These results suggest that Val at this site plays a role in determining the interaction of the HIV-1 RT enzyme with the pyrophosphate group of deoxynucleoside triphosphate (dNTP) and that the hydrophobicity of the amino acid at this position was the most important determinant in the binding of HIV-1 RT to dNTP.
Im
G J
GJ
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
Tramontano
E
E
Gonzalez
C J
CJ
Cheng
Y C
YC
D000596:Amino Acids / Q000378:metabolism*; D000998:Antiviral Agents / Q000378:metabolism*; D001665:Binding Sites; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D017245:Foscarnet / Q000378:metabolism*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism
D000596:Amino Acids; D000998:Antiviral Agents; D054306:Dideoxynucleotides; D011756:Diphosphates; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D017245:Foscarnet; D015215:Zidovudine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase