T5核酸外切酶水解乙型肝炎病毒复制中间体允许通过PCR可靠地

StephenW

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
Bingqian Qu, Yi Ni, Florian A. Lempp, Florian W. R. Vondran, Stephan Urban
J.-H. James Ou, Editor
DOI: 10.1128/JVI.01117-18

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ABSTRACT

Chronic infection with the human hepatitis B virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular DNA (cccDNA), the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular DNA [rcDNA]), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and Southern blotting or selective quantitative PCR (qPCR) methods allowing discrimination of cccDNA and rcDNA. In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases for targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximately 1-kb fragment permits reliable quantification of cccDNA without the requirement of prior nucleus enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicities of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance.

IMPORTANCE cccDNA elimination is a major goal in future curative regimens for chronic HBV patients. However, PCR-based assays for cccDNA quantification show a principally constrained specificity when high levels of input virus or replicative intermediates are present. Here, we characterized T5 exonuclease as a suitable enzyme for medium-throughput in vitro assays that preserves cccDNA but efficiently removes rcDNA prior to PCR-based quantification. We compared T5 exonuclease with the previously described exonuclease III and showed that both nucleases are suitable for reliable quantification of cccDNA by PCR. We substantiated the applicability of our method through examination of early cccDNA formation and stable accumulation in several in vitro infection models and analyzed cccDNA stability after administration of anti-HBV drugs. Our results support the use of T5 exonuclease for fast and convenient rcDNA removal, especially for early cccDNA quantification and rapid drug testing in in vitro studies.

    Copyright © 2018 American Society for Microbiology.

StephenW

T5核酸外切酶水解乙型肝炎病毒复制中间体允许通过PCR可靠地定量和快速药物功效测试共价闭合环状DNA
Bingqian Qu，Yi Ni，Florian A. Lempp，Florian W. R. Vondran，Stephan Urban
J.-H. James Ou，编辑
DOI：10.1128 / JVI.01117-18

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抽象

慢性感染人类乙型肝炎病毒（HBV）是一个主要的健康问题。病毒持续性需要建立和维持共价闭合环状DNA（cccDNA），即感染肝细胞核中的附加型病毒模板。与复制DNA中间体（松弛环状DNA [rcDNA]）相比，感染的肝细胞中cccDNA的拷贝数较低。因此，cccDNA的准确分析需要富集核部分和Southern印迹或选择性定量PCR（qPCR）方法，以允许区分cccDNA和rcDNA。在本报告中，我们分析了cccDNA特异性引物对选择性扩增cccDNA的能力。使用确定形式的HBV和基因组DNA的混合物，我们确定了不同核酸酶在不存在和存在基因组DNA的情况下靶向消化开放/松弛环状DNA形式而不影响cccDNA的潜力。我们发现T5核酸外切酶与扩增约1-kb片段的引物组合的组合允许可靠地定量cccDNA而无需先前的细胞核富集或Hirt提取。我们在四种不同的体外感染系统中测试了该方法，并且在接种的基因组当量的多重性增加时量化了cccDNA拷贝数。我们进一步分析了cccDNA形成的动力学以及药物（干扰素，进入抑制剂和衣壳抑制剂）对cccDNA的影响。我们的方法允许在存在高过量输入病毒和复制中间体的情况下在感染的早期阶段进行可靠的cccDNA定量，从而适用于药物筛选和cccDNA形成和维持的研究。

重要性cccDNA消除是慢性HBV患者未来治疗方案的主要目标。然而，当存在高水平的输入病毒或复制中间体时，用于cccDNA定量的基于PCR的测定显示出主要受限的特异性。在这里，我们将T5外切核酸酶鉴定为适合于中等通量体外测定的酶，其保留cccDNA但在基于PCR的定量之前有效去除rcDNA。我们比较了T5核酸外切酶与之前描述的核酸外切酶III，并显示两种核酸酶都适合通过PCR可靠地定量cccDNA。我们通过检测早期cccDNA形成和几种体外感染模型中的稳定积累来证实了我们的方法的适用性，并分析了抗HBV药物给药后的cccDNA稳定性。我们的结果支持使用T5核酸外切酶快速方便地去除rcDNA，尤其是早期cccDNA定量和体外研究中的快速药物测试。

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