T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR
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DOI：
10.1128/jvi.01117-18
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发表时间：
2018-12-01
影响因子：
5.4
通讯作者：
Urban, Stephan
中科院分区：
文献类型：
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作者：
Qu, Bingqian;Ni, Yi;Urban, Stephan
人类B型肝炎病毒（HBV）的慢性感染是一个主要的健康问题。病毒的持续存在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体病毒模板。与复制型DNA中间体（松弛环状DNA [rcDNA]）相比，感染肝细胞中cccDNA的拷贝数较低。因此，cccDNA的准确分析需要富集核级分和Southern印迹或选择性定量PCR（qPCR）方法，从而允许区分cccDNA和rcDNA。在这份报告中，我们分析了cccDNA特异性引物对选择性扩增cccDNA的能力。使用定义形式的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片段的引物组的组合允许可靠地定量cccDNA，而不需要预先进行核富集或Hirt提取。我们在四种不同的体外感染系统中测试了这种方法，并在接种基因组当量的多重性增加时定量cccDNA拷贝数。我们进一步分析了cccDNA形成的动力学和药物（干扰素、进入抑制剂和衣壳抑制剂）对cccDNA的影响。我们的方法允许在感染的早期阶段在输入病毒和复制中间体的存在下进行可靠的cccDNA定量，因此适用于药物筛选和cccDNA形成和维持的研究。重要信息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，基于PCR的cccDNA定量分析显示出主要受限的特异性。在这里，我们将T5核酸外切酶表征为用于中等通量体外测定的合适酶，其保留cccDNA但在基于PCR的定量之前有效地去除rcDNA。我们将T5核酸外切酶与先前描述的核酸外切酶III进行了比较，并表明这两种核酸酶都适用于通过PCR对cccDNA进行可靠的定量。我们通过在几种体外感染模型中检查早期cccDNA形成和稳定积累并分析抗HBV药物给药后cccDNA的稳定性来证实我们方法的适用性。我们的研究结果支持使用T5核酸外切酶快速方便地去除rcDNA，特别是用于体外研究中的早期cccDNA定量和快速药物检测。
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.