Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine (AZT):: Increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase
Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine (AZT):: Increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase
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DOI：
10.1021/bi981200e
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发表时间：
1998-11-10
期刊：
影响因子：
2.9
通讯作者：
Parniak, MA
中科院分区：
文献类型：
--
作者：
Arion, D;Kaushik, N;Parniak, MA
与AZT高水平耐药相关的多重突变（D67N, K70R, T215F, K219Q）出现在病毒逆转录酶（RT）的两个不同亚域，表明这些突变可能对总体耐药有不同的贡献。我们将野生型RT与D67N/K70R/T215F/K219Q、D67N/K70R和T215F/K219Q突变酶进行了比较。D67N/K70R/T215F/K219Q突变体DNA聚合酶表达能力增强；这是由于模板/引物与RT的分离减少，并且是由于T215F/K219Q突变。在0.5 mM焦磷酸盐存在下，D67N/K70R/T215F/K219Q突变体对AZTTP的敏感性（IC50约为300 nM）低于wt RT （IC50 × 100 nM）。这种焦磷酸盐介导的突变酶敏感性的变化对AZTTP是选择性的，因为在没有或存在焦磷酸盐的情况下，wt和突变体RT对TTP的K-m值和ddCTP和ddGTP的抑制作用相似。D67N/K70R/T215F/K219Q突变体显示端链DNA的热磷酸化（DNA合成的逆反应）速率增加；这种增强的焦磷分解是由于D67N/K70R突变。然而，对于野生型和突变型酶，热磷酸化的过程是相似的。我们认为HIV-1对AZT的抗性是由于突变体RT在生理焦磷酸盐水平上选择性地减少了AZTTP的结合和增加了对链终止病毒DNA的焦磷裂解裂解，导致链终止的净减少。通过补偿逆反应速率的增加，病毒DNA合成能力的增加可能对在AZT存在下使HIV易于复制很重要。
The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximate to 300 nM) than wt RT (IC50 x 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar K-m values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.