Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.
German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
J Virol. 2018 Nov 12;92(23). doi: 10.1128/JVI.01117-18. Print 2018 Dec 1.
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 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. 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 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 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 studies.
慢性乙型肝炎病毒（HBV）感染是一个主要的健康问题。病毒持续存在需要建立和维持共价闭合环状 DNA（cccDNA），cccDNA 是感染肝细胞中病毒的附加体模板。与复制性 DNA 中间体（松弛环状 DNA [rcDNA]）相比，感染肝细胞中的 cccDNA 拷贝数较低。因此，cccDNA 的准确分析需要富集核部分并进行 Southern 印迹或选择性定量 PCR（qPCR）方法，以区分 cccDNA 和 rcDNA。在本报告中，我们分析了用于选择性扩增 cccDNA 的特异性引物对。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 时靶向消化开放/松弛环状 DNA 形式的潜力，而不影响 cccDNA。我们发现，T5 外切核酸酶与扩增大约 1kb 片段的引物对的组合允许在无需预先富集核或 Hirt 提取的情况下可靠地定量 cccDNA。我们在四个不同的 感染系统中测试了这种方法，并在递增接种基因组当量的倍数时定量了 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大量输入病毒和复制中间体的情况下，在感染的早期阶段可靠地定量 cccDNA，并且适合药物筛选和 cccDNA 形成和维持的研究。cccDNA 的消除是慢性 HBV 患者未来治愈方案的主要目标。然而，基于 PCR 的 cccDNA 定量检测在存在高水平输入病毒或复制中间体时表现出本质上的限制特异性。在这里，我们将 T5 外切核酸酶描述为一种适合中等通量 检测的合适酶，它可以在 PCR 定量之前保留 cccDNA，但有效地去除 rcDNA。我们比较了 T5 外切核酸酶和之前描述的外切核酸酶 III，并表明这两种核酸酶都适合通过 PCR 可靠地定量 cccDNA。我们通过在几种 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，证明了我们方法的适用性。我们的结果支持使用 T5 外切核酸酶快速方便地去除 rcDNA，特别是用于早期 cccDNA 定量和快速药物检测 研究。
