Sensitivity of HIV-1 reverse transcriptase and its mutants to inhibition by azidothymidine triphosphate.
HIV-1 reverse transcriptase can catalyze the addition of either azidothymidine monophosphate (AZTMP) or thymidine monophosphate (dTMP) to a primer strand opposite template adenosine bases. The ratio of incorporation of AZTMP to dTMP as catalyzed by HIV-1 reverse transcriptase has been determined to be 0.4 using an RNA-DNA duplex substrate prepared from oligonucleotides with sequences taken from the HIV-1 genome sequence. Slight variations are found for the incorporation ratio of the two nucleotides on other substrates. Substrates containing more than one adenosine in the single-stranded part of the template allow for more chances to incorporate AZTMP and less full-length product. Variations in the intensity of bands on an autoradiograph of a DNA sequencing gel corresponding to different positions of incorporation of AZTMP suggest that not all template adenosine positions offer the same level of discrimination against incorporation of AZTMP. A reverse transcriptase containing a set of four mutations (D67N, K70R, T215Y, K219Q) known to cause resistance to AZT in cell culture assays has a ratio of incorporation that is 0.77 +/- 0.03 times the ratio for the wild-type reverse transcriptase opposite one specific template adenosine. In contrast, a hybrid mutant containing the same four mutations that cause resistance to AZT and an additional mutation, Y181C, which by itself causes resistance to the non-nucleoside inhibitor L-697,661 [Sardana et al. (1992), J. Biol. Chem. 267, 17526-17530], has a ratio of incorporation that is 1.34 +/- 0.01 times that of the wild-type, indicating that the hybrid mutant enzyme is more susceptible to inhibition by AZTTP than the wild-type reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)
Carroll
S S
SS
Department of Biological Chemistry, Merck Research Labs, West Point, Pennsylvania 19486.
Geib
J
J
Olsen
D B
DB
Stahlhut
M
M
Shafer
J A
JA
Kuo
L C
LC
D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity; D013698:Templates, Genetic; D013938:Thymidine Monophosphate / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D000998:Antiviral Agents; D017931:DNA Primers; D054306:Dideoxynucleotides; D011756:Diphosphates; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D013938:Thymidine Monophosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase