Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase
Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase
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DOI：
10.1128/jvi.02575-13
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发表时间：
2014-03-01
影响因子：
5.4
通讯作者：
Ferguson, Neil
中科院分区：
文献类型：
--
作者：
Voeroes, Judit;Urbanek, Annika;Ferguson, Neil
乙型肝炎病毒 (HBV) 是一种主要的人类病原体，每年导致严重的肝脏疾病和 60 万人死亡。已批准的治疗慢性 HBV 感染的疗法通常以多功能病毒聚合酶 (hPOL) 为目标。不幸的是，这些疗法——广谱抗病毒药物——不是一般疗法，有副作用，并会导致病毒耐药。虽然 hPOL 仍然是一个有吸引力的治疗靶点，但众所周知，它很难以可溶形式以适合结构研究的产量表达和纯化。因此，hPOL 不存在经验结构数据，这阻碍了针对 HBV 的药物化学和合理的先导化合物发现工作。在这里，我们提出了一种在大肠杆菌中过表达重组 hPOL 结构域的有效策略，以高产率纯化它们并解决其已知的聚集倾向。这使我们能够对 hPOL 结构域进行首次结构和生物物理表征。 Apo-hPOL 结构域主要采用 α 螺旋结构，含有少量 β 折叠结构。我们的重组材料在体外表现出金属依赖性逆转录酶活性，金属结合调节 hPOL 结构。 Calcomine Orange 2RS 是一种抑制鸭 HBV POL 活性的小分子，也抑制重组 hPOL 的体外引发活性。我们的工作为 hPOL 的结构和生物物理表征铺平了道路，并应有助于 HBV 的高通量先导物发现。重要性来自人乙型肝炎病毒 (hPOL) 的病毒聚合酶是一个经过充分验证的治疗靶点。然而，重组 hPOL 因其极难以可溶性活性形式表达且产量适合通常在药物发现项目中发挥重要作用的结构研究而享有盛誉。这阻碍了急需的新型乙肝病毒抗病毒药物的开发。然而，我们已经解决了这个问题，并在此报告了在大肠杆菌中表达重组 hPOL 结构域的程序，以及以具有体外活性的可溶形式纯化它们的方法。我们还首次展示了 hPOL 的结构和生物物理特征。我们的工作为 hPOL 结构和功能的新见解铺平了道路，这将有助于发现新型 HBV 抗病毒药物。
Hepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies-broad-spectrum antivirals-are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly alpha-helical structures with small amounts of beta-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV.IMPORTANCEThe viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.