通过聚合酶反应解开乙型肝炎病毒松弛环状 DNA 为共价闭合环

StephenW

通过聚合酶反应解开乙型肝炎病毒松弛环状 DNA 为共价闭合环状 DNA 的准确定量和可视化提供了新的选择
Naohiro Kamiya 1 2 , Takahiko Sugimoto 1 , Hiromi Abe-Chayama 2 3 , Rie Akiyama 2 3 , Yasunori Tsuboi 1 , Akira Mogami 1 , Michio Imamura 2 3 , C Nelson Hayes 2 3 , Kazuaki Chayama 2 3 4
隶属关系
隶属关系

    1
    研究单位/免疫学和炎症，Sohyaku，创新研究部，三菱田边制药公司，横滨，神奈川县，日本。
    2
    日本广岛大学生物医学与健康科学研究生院胃肠病学和代谢学系。
    3
    广岛大学肝病学和胃肠病学研究中心，日本广岛。
    4
    日本横滨理化研究所 (RIKEN) 综合医学科学中心。

    PMID：35130138 DOI：10.1099/jgv.0.001591

抽象的

乙型肝炎病毒 (HBV) 是一种小型嗜肝 DNA 病毒，通过 RNA 中间体进行复制。进入后，病毒衣壳携带松弛的环状 DNA (rcDNA) 进入细胞核，在那里病毒基因组被转化为共价闭合环状 DNA (cccDNA)，作为所有病毒转录物的模板。为了监测 cccDNA 水平，已经出现了用于定量 PCR 的消除 rcDNA 的预处理方法，尽管 Southern 印迹仍然是区分 cccDNA 与其他 DNA 中间体的唯一方法。在这项研究中，我们建立了一种使用特定聚合酶将成熟 rcDNA 解开成双链线性 DNA 的稳健方法。 Untying rcDNA 不仅为 cccDNA 定量提供了一种替代方法，而且为可视化 cccDNA 提供了一种灵敏的方法。我们将这种方法与质粒安全 DNase 和 T5 核酸外切酶预处理相结合，发现准确的定量需要通过限制性内切酶消化 cccDNA，因为 T5 核酸外切酶处理后 cccDNA 的热稳定性增加。在使用双链 TaqMan 探针的数字 PCR 中，少于 1000 个 cccDNA 拷贝被成功地可视化为双阳性点，这与来自未绑定 rcDNA 的单阳性不同。该方法进一步应用于用核苷类似物和核心蛋白变构调节剂处理的原代肝细胞感染模型，以监测 cccDNA 水平。通过人类基因组拷贝对 cccDNA 的相对定量证明了精确评估每个细胞核的 cccDNA 水平的可能性。这些结果清楚地表明，解开 rcDNA 的顺序反应可用于研究一小部分细胞核中的 cccDNA 命运。

关键词：cccDNA；数字PCR；乙型肝炎病毒;量化。

StephenW

Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA
Naohiro Kamiya  1   2 , Takahiko Sugimoto  1 , Hiromi Abe-Chayama  2   3 , Rie Akiyama  2   3 , Yasunori Tsuboi  1 , Akira Mogami  1 , Michio Imamura  2   3 , C Nelson Hayes  2   3 , Kazuaki Chayama  2   3   4
Affiliations
Affiliations

    1
    Research Unit/Immunology & Inflammation, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Kanagawa, Japan.
    2
    Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
    3
    Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan.
    4
    Institute of Physical and Chemical Research (RIKEN) Center for Integrative Medical Sciences, Yokohama, Japan.

    PMID: 35130138 DOI: 10.1099/jgv.0.001591

Abstract

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Keywords: cccDNA; digital PCR; hepatitis B virus; quantification.