SENSITIVITY OF HIV-1 REVERSE-TRANSCRIPTASE AND ITS MUTANTS TO INHIBITION BY AZIDOTHYMIDINE TRIPHOSPHATE
SENSITIVITY OF HIV-1 REVERSE-TRANSCRIPTASE AND ITS MUTANTS TO INHIBITION BY AZIDOTHYMIDINE TRIPHOSPHATE
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DOI：
10.1021/bi00174a018
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发表时间：
1994-03-01
期刊：
影响因子：
2.9
通讯作者：
KUO, LC
中科院分区：
文献类型：
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作者：
CARROLL, SS;GEIB, J;KUO, LC
HIV-1逆转录酶可以催化叠氮胸苷一磷酸（AZTMP）或胸苷一磷酸（dTMP）与模板腺苷碱基相对的引物链加成。使用由具有取自HIV-1基因组序列的序列的寡核苷酸制备的RNA-DNA双链体底物，已确定HIV-1逆转录酶催化的AZTMP与dTMP的掺入比为0.4。发现两种核苷酸在其他底物上的掺入比略有变化。在模板的单链部分中含有多于一个腺苷的底物允许更多的机会掺入AZTMP和更少的全长产物。在DNA测序凝胶的放射自显影上对应于AZTMP掺入的不同位置的条带强度的变化表明，并非所有模板腺苷位置都提供相同水平的对AZTMP掺入的区分。在细胞培养试验中，已知含有一组四种突变（D 67 N、K70 R、T215 Y、K219 Q）的逆转录酶可引起AZT耐药性，其掺入比是野生型逆转录酶与一种特异性模板腺苷的掺入比的0.77 +/-0.03倍。相反，含有引起对AZT的抗性的相同的四个突变和另外的突变Y181 C的杂合突变体，Y181 C本身引起对非核苷抑制剂L-697，661的抗性[Sardana等（1992），J.Biol.Chem.267，17526-17530]，具有的掺入比是野生型的1.34 +/-0.01倍，表明杂合突变酶比野生型逆转录酶更容易受到AZTTP的抑制。HIV-1逆转录酶能够在生理浓度的焦磷酸盐存在下催化除去掺入的AZTMP。然而，从AZTMP终止的引物中去除AZTMP的二级速率常数对于野生型逆转录酶仅为3.4 M（-1）s（-1），对于AZT抗性形式的逆转录酶仅为1.5 M（-1）s（-1）。已经确定了许多其他位点特异性突变体（对核苷或非核苷抑制剂具有抗性）掺入AZTMP的能力。
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. HIV-1 reverse transcriptase is able to catalyze removal of incorporated AZTMP in the presence of physiological concentrations of pyrophosphate. The second-order rate constant for removal of AZTMP from AZTMP-terminated primers is, however, only 3.4 M(-1) s(-1) for the wild-type reverse transcriptase and 1.5 M(-1) s(-1) for the AZT-resistant form of reverse transcriptase. The abilities of a number of other site-specific mutants, resistant to either nucleoside or non-nucleoside inhibitors, to incorporate AZTMP have been determined.