Initial binding of the broad spectrum antiviral nucleoside ribavirin to the hepatitis C virus RNA polymerase
Initial binding of the broad spectrum antiviral nucleoside ribavirin to the hepatitis C virus RNA polymerase
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DOI：
10.1074/jbc.m308917200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Bisaillon, M
中科院分区：
文献类型：
--
作者：
Bougie, I;Bisaillon, M
利巴韦林是一种广谱抗病毒核苷，对多种RNA和DNA病毒显示出活性。利巴韦林目前与干扰素-α联合用于治疗丙型肝炎病毒（HCV）感染，最近显示可通过HCV RNA聚合酶直接掺入RNA产物中。这种能力最终导致突变率增加，并大大降低了病毒的适应性。作为阐明利巴韦林和HCV聚合酶之间特异性相互作用性质的第一步，我们利用荧光光谱法精确监测三磷酸利巴韦林（RTP）与病毒聚合酶的结合。这种光谱方法使我们能够清楚地将RTP结合活性与伴随的催化步骤分开。我们在这里报告的第一个详细的研究RTP和RNA聚合酶之间的相互作用的结合动力学和热力学参数。我们证明，RTP绑定到相同的活性位点的核苷酸。此外，我们提供的证据表明，丙型肝炎病毒聚合酶不仅可以结合RTP，但也非磷酸化利巴韦林，虽然具有较低的亲和力。通过使用模板引物的各种组合，我们还证明了碱基配对不参与RTP与HCV聚合酶的初始结合。基于圆二色谱和变性研究的结果，我们表明，RNA聚合酶的RTP的结合后，经历了微妙的构象变化，虽然相互作用并没有显着修改的蛋白质的稳定性。最后，虽然金属离子是催化活性所必需的，但它们不是RTP与聚合酶的初始结合所必需的。这种定量分析是最重要的合理设计的新的利巴韦林类似物的潜在治疗价值，并提供了重要的见解RTP和HCV RNA聚合酶之间的相互作用。
Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-alpha for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.