Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.
Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.
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DOI：
10.1074/jbc.m117.798256
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发表时间：
2018-01-05
期刊：
影响因子：
--
通讯作者：
Nowotny M
中科院分区：
文献类型：
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作者：
Figiel M;Krepl M;Park S;Poznański J;Skowronek K;Gołąb A;Ha T;Šponer J;Nowotny M
HIV-1逆转录酶(RT)具有DNA聚合酶活性和核糖核酸酶H活性，两者协同作用，将病毒基因组的单链RNA转化为双链DNA，然后整合到感染细胞的DNA中。逆转录酶催化的逆转录在很大程度上依赖于多尿路(PPT)引物的正确产生。然而，PPT引物的产生机制和PPT序列的特征对于其被HIV-1 RT识别是至关重要的，目前仍不清楚。在这里，我们使用化学交联法，结合分子动力学模拟和单分子检测来研究PPT引物的产生机理。我们发现，PPT在共价连接的HIV-1RT-核酸复合体中被特异性和正确地识别。这些发现表明，PPT在形成后在稳定的催化络合物中发生识别。我们发现，这种独特的识别是基于两个依赖于PPT序列的互补元件：RNaseH序列偏好和PPT的聚(Ra/dt)链与RNaseH裂解所需的核酸构象不相容。后者是由于聚(Ra/dt)链的刚性造成的，并导致该序列在变形成催化相关的几何构型时发生碱基对滑移。综上所述，我们的结果揭示了一种基于聚(Ra/dt)片段特定动态特性的PPT引物产生的意想不到的机制，并有助于我们对病毒RNA反转录机制的理解。
HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase–catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT–nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.