乙型肝炎病毒复制中间体的T5核酸外切酶水解允许通过PCR对共

StephenW

J Virol. 2018 Sep 19. pii: JVI.01117-18. doi: 10.1128/JVI.01117-18. [Epub ahead of print]
T5 exonuclease hydrolysis of Hepatitis B Virus replicative intermediates allows reliable quantification and fast drug efficacy testing of covalently closed circular DNA by PCR.
Qu B1, Ni Y1,2, Lempp FA1,2, Vondran FWR3,4, Urban S5,2.
Author information

1
    Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.
2
    German Centre for Infection Research (DZIF), Partner Site Heidelberg, Germany.
3
    Regenerative Medicine and Experimental Surgery (ReMediES), Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hannover, Germany.
4
    German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany.
5
    Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany [email protected].

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 (ccc) DNA, the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular (rc) DNA), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and southern blotting or selective 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 to 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 approximate 1 kb fragment permits reliable quantification of cccDNA without the requirement of prior nuclei enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicity 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.ImportancecccDNA 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 show 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.

PMID:
    30232183
DOI:
    10.1128/JVI.01117-18

StephenW

J Virol。 2018年9月19日.pii：JVI.01117-18。 doi：10.1128 / JVI.01117-18。 [提前打印]
乙型肝炎病毒复制中间体的T5核酸外切酶水解允许通过PCR对共价闭合环状DNA进行可靠的定量和快速药物功效测试。
Qu B1，Ni Y1,2，Lempp FA1,2，Vondran FWR3,4，Urban S5,2。
作者信息

1
    德国海德堡海德堡大学医院传染病科，分子病毒学系。
2
    德国感染研究中心（DZIF），德国海德堡合作伙伴网站。
3
    再生医学和实验外科（ReMediES），德国汉诺威汉诺威医学院普通，内脏和移植外科。
4
    德国感染研究中心（DZIF），合作伙伴网站Hannover-Braunschweig，德国。
五
    海德堡大学医院分子病毒学传染病系，德国海德堡，Stephan.Urban @med.uni-heidelberg.de。

抽象

人类乙型肝炎病毒（HBV）的慢性感染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（ccc）DNA，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（rc）DNA）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qPCR方法，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外切核酸酶与扩增大约1kb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样性增加时量化了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。我们的方法允许在高过量输入病毒和复制中间体存在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。重要的cccDNA消除是未来治疗方案的主要目标。慢性HBV患者。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，并表明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，特别是在体外研究中用于早期cccDNA定量和快速药物测试。

结论：
    30232183
DOI：
    10.1128 / JVI.01117-18

齐欢畅

好文章。
其实我在想就基因编辑技术来说，如果我们可以找到一种编辑技术，只是针对cccdna的，就可以大大的降低病毒。
现在的基因编辑技术确实还有不稳定现象，顾虑。
但是如果只是针对cccdna呢，破坏掉cccdna就可以了，人体内的整合dna不要去碰，因为整合dna有可能引起人的细胞正常基因或染色体 的问题。
其实如果能解决ccdna的问题，那也已经是很大进步了。