Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication
Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication
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DOI：
10.1074/jbc.m604460200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Burke, Donald H.
中科院分区：
文献类型：
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作者：
Held, Daniel M.;Kissel, Jay D.;Burke, Donald H.
HIV-1逆转录酶（RT）的核酸适体是体外DNA聚合酶功能的有效抑制剂，并且当在培养的T淋巴细胞系中表达时，它们已被证明能抑制病毒复制。我们监测RT抑制五RNA假结RNA适体在一系列的生化试验，旨在模拟病毒逆转录的离散步骤。我们的研究结果表明，所有RT功能测定，包括RNA和DNA引发的DNA聚合，链置换合成，和聚合酶独立的RNase H活性的适体抑制（IC 50值在低纳摩尔范围内）。此外，我们观察到适体抑制的时间依赖性的差异。聚合酶非依赖性RNA酶H活性对长期适体抑制最具抗性，而RNA依赖性DNA聚合最易受影响。最后，当在RNA适体与四种不同的小分子抑制剂中的每一种组合的存在下监测DNA聚合时，在适体和两种核苷类似物RT抑制剂（叠氮胸苷三磷酸或ddCTP）之间观察到显著的协同作用，而两种非核苷类似物RT抑制剂显示弱协同作用（依法韦仑）或拮抗作用（奈韦拉平）。总之，这些结果支持一种模型，其中适体通过在基因组复制的每个阶段累积抑制RT来抑制病毒复制。
Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA- dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.