规模化生产和人类乙型肝炎病毒聚合酶的结构和生物物理表

StephenW

Large-Scale Production and Structural and Biophysical Characterizations of the Human Hepatitis B Virus Polymerase

    Judit Vörösa,
    Annika Urbanekb,
    Gilles Jean Philippe Rautureaua*,
    Maggie O'Connorb,
    Henry C. Fisherc,
    Alison E. Ashcroftc and
    Neil Fergusonb

    a School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland
    b School of Medicine and Medical Science, University College Dublin, Dublin, Ireland
    c Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom

    D. S. Lyles, Editor

+ Author Affiliations
ABSTRACT

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 α-helical structures with small amounts of β-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.

IMPORTANCE The 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.
FOOTNOTES

        Received 5 September 2013.
        Accepted 9 December 2013.
    Address correspondence to Neil Ferguson, [email protected].

    J.V., A.U., and G.J.P.R. contributed equally to this work.

    ↵* Present address: Gilles Jean Philippe Rautureau, Ecole Normale Supérieure de Lyon, Centre de RMN à Très Hauts Champs, UMR 5280 CNRS/ENS, Villeurbanne, France.

    Published ahead of print 18 December 2013

    Copyright © 2014, American Society for Microbiology. All Rights Reserved.

StephenW

规模化生产和人类乙型肝炎病毒聚合酶的结构和生物物理表征

    尤迪特Vörösa ，
    安妮卡Urbanekb ，
    吉尔斯·吉恩菲利普Rautureaua * ，
    张曼玉O'Connorb ，
    亨利C Fisherc ，
    艾莉森E. Ashcroftc和
    尼尔Fergusonb

    分子生物和生物医学科学，都柏林大学，都柏林，爱尔兰aSchool
    医学bSchool和医学科学，都柏林大学，爱尔兰都柏林
    利兹，利兹大学cAstbury中心结构分子生物学，英国

    D. S.莱尔斯，编辑器

+作者所属机构
摘要

乙型肝炎病毒（HBV ）是一种主要的人类病原体，导致严重的肝脏疾病，每年60万人死亡。批准的疗法用于治疗慢性HBV感染通常针对多功能病毒聚合酶（ HPOL ） 。不幸的是，这些疗法广谱抗病毒剂，是不一般的治疗方法，有副作用，并且会导致病毒抗性。而HPOL仍然是一个有吸引力的治疗靶标，这是非常困难的表达，并在适当的用于结构研究收率纯化的可溶形式。因此，对于HPOL没有经验结构数据存在，这妨碍了药物化学和乙肝病毒靶向理性先导化合物的发现工作。在这里，我们提出了一个有效的策略，以过度的重组HPOL域在大肠杆菌中，净化他们在高产量和解决他们已知的聚集倾向。这使我们能够执行HPOL域的第一结构和生物物理表征。 APO- HPOL域采用主要有α -螺旋结构与少量β-折叠结构。我们的重组体材料表现出金属依赖性，逆转录酶的活性在体外，与金属结合的调制HPOL结构。 CALCOMINE橙2RS，一个小分子，抑制鸭HBV POL活性，也抑制了重组HPOL的体外引发活性。我们的工作铺平了道路HPOL的结构和生物物理表征，并应促进高通量先导化合物的发现为乙肝病毒。

重要性病毒聚合酶从人类B型肝炎病毒（ HPOL ）是一种经过验证的治疗靶点。然而，重组HPOL有一个被极其困难的可溶性，活性形式来表达适当的结构研究，平时玩在药物发现计划的重要作用，收益率名不虚传。这阻碍了对乙肝病毒急需的新的抗病毒药物的开发。然而，我们已解决了这个问题，在这里报告程序，用于表达重组HPOL结构域在大肠杆菌和也用于纯化它们在具有活性的体外可溶形式的方法。我们还提出HPOL的第一结构和生物物理表征。我们的工作铺平了道路，新的见解HPOL结构和功能，这应有助于新型抗病毒药物的发现为乙肝病毒。
脚注

        收到2013年9月5日。
        接受2013年12月9日。
    地址对应尼尔·弗格森， [email protected] 。

    J.V. ， A.U.和G.J.P.R.同等贡献这项工作。

    ↵ *现住址：吉尔斯·吉恩菲利普Rautureau ，高等师范学院里昂，中心德RMN单TRES HAUTS香榭丽舍， UMR 5280 CNRS / ENS ，法国Villeurbanne 。

    提前出版印刷二○一三年十二月一十八日

    ©版权所有2014年，美国微生物学会。保留所有权利。