前基因组 RNA 启动乙型肝炎病毒复制系统有助于抗病毒剂和耐

StephenW

前基因组 RNA 启动乙型肝炎病毒复制系统有助于抗病毒剂和耐药变体对共价闭合环状 DNA 合成的机理研究
Qiong Zhao 1、Jinhong Chang 1、Rene Rijnbrand 2、Angela M Lam 2、Michael J Sofia 2、Andrea Cuconati 2、郭巨涛 1
隶属关系
隶属关系

    1个
    Baruch S. Blumberg 研究所，美国宾夕法尼亚州多伊尔斯敦。
    2个
    Arbutus Biopharma, Inc.，美国宾夕法尼亚州沃明斯特。

    PMID：36448800 DOI：10.1128/jvi.01150-22

抽象的

乙型肝炎病毒 (HBV) 通过在核衣壳内逆转录 RNA 中间体（称为前基因组 RNA (pgRNA)）来复制其基因组 DNA。已经表明，体外转录的 pgRNA 的转染启动了人类肝癌细胞中的病毒复制。我们在此证明，病毒衣壳、单链 DNA、松弛环状 DNA (rcDNA) 和共价闭合环状 DNA (cccDNA) 在 pgRNA 转染到 Huh7.5 细胞后 3、6、12 和 24 小时可以连续检测到。病毒 DNA 复制中间体和 cccDNA 的水平分别在 pgRNA 转染后 24 小时和 48 小时达到峰值。转染后第 4 天，培养基中可检测到 HBV 表面抗原 (HBsAg)。有趣的是，早期强大的病毒 DNA 复制和 cccDNA 合成不依赖于 HBV X 蛋白 (HBx) 的表达，而 HBsAg 的产生严格依赖于病毒 DNA 复制和 HBx 的表达，这与 HBx 在转录过程中的重要作用一致cccDNA 微型染色体的激活。虽然 pgRNA 转染后 48 小时内强大且同步的 HBV 复制特别适用于 HBV 复制步骤的精确映射，从衣壳组装到 cccDNA 形成，由不同的抗病毒剂靶向，但细胞处理从转染后 48 小时开始pgRNA 转染允许评估抗病毒剂对成熟核衣壳脱壳、cccDNA 合成和转录以及病毒 RNA 稳定性的影响。此外，pgRNA 发射系统可用于轻松评估耐药变体对 cccDNA 形成和病毒生命周期中其他复制步骤的影响。重要性 肝炎病毒 pgRNA 不仅作为病毒 DNA 逆转录复制的模板，还表达核心蛋白和 DNA 聚合酶以支持病毒基因组复制和 cccDNA 合成。毫不奇怪，鸭乙型肝炎病毒 pgRNA 的细胞质表达启动了病毒复制，导致感染性病毒体分泌。然而，HBV 复制和抗病毒机制主要是在用基于质粒的 HBV 复制子瞬时或稳定转染的人肝癌细胞中研究的。细胞染色体中大量转染的 HBV DNA 或转基因的存在阻碍了对 HBV 复制和 cccDNA 功能的稳健分析。如此处所示，pgRNA 发射 HBV 复制系统允许使用分泌的 HBsAg 作为方便的定量标记准确定位抗病毒靶标和研究 cccDNA 生物合成和转录。耐药变体对病毒衣壳组装、基因组复制以及 cccDNA 生物合成和功能的影响也可以使用该系统进行评估。

关键词：抗病毒药物；衣壳组装；衣壳拆卸；乙型肝炎病毒;病毒复制。

StephenW

Pregenomic RNA Launch Hepatitis B Virus Replication System Facilitates the Mechanistic Study of Antiviral Agents and Drug-Resistant Variants on Covalently Closed Circular DNA Synthesis
Qiong Zhao  1 , Jinhong Chang  1 , Rene Rijnbrand  2 , Angela M Lam  2 , Michael J Sofia  2 , Andrea Cuconati  2 , Ju-Tao Guo  1
Affiliations
Affiliations

    1
    Baruch S. Blumberg Institute, Doylestown, Pennsylvania, USA.
    2
    Arbutus Biopharma, Inc., Warminster, Pennsylvania, USA.

    PMID: 36448800 DOI: 10.1128/jvi.01150-22

Abstract

Hepatitis B virus (HBV) replicates its genomic DNA by reverse transcription of an RNA intermediate, termed pregenomic RNA (pgRNA), within nucleocapsid. It had been shown that transfection of in vitro-transcribed pgRNA initiated viral replication in human hepatoma cells. We demonstrated here that viral capsids, single-stranded DNA, relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA) became detectable sequentially at 3, 6, 12, and 24 h post-pgRNA transfection into Huh7.5 cells. The levels of viral DNA replication intermediates and cccDNA peaked at 24 and 48 h post-pgRNA transfection, respectively. HBV surface antigen (HBsAg) became detectable in culture medium at day 4 posttransfection. Interestingly, the early robust viral DNA replication and cccDNA synthesis did not depend on the expression of HBV X protein (HBx), whereas HBsAg production was strictly dependent on viral DNA replication and expression of HBx, consistent with the essential role of HBx in the transcriptional activation of cccDNA minichromosomes. While the robust and synchronized HBV replication within 48 h post-pgRNA transfection is particularly suitable for the precise mapping of the HBV replication steps, from capsid assembly to cccDNA formation, targeted by distinct antiviral agents, the treatment of cells starting at 48 h post-pgRNA transfection allows the assessment of antiviral agents on mature nucleocapsid uncoating, cccDNA synthesis, and transcription, as well as viral RNA stability. Moreover, the pgRNA launch system could be used to readily assess the impacts of drug-resistant variants on cccDNA formation and other replication steps in the viral life cycle. IMPORTANCE Hepadnaviral pgRNA not only serves as a template for reverse transcriptional replication of viral DNA but also expresses core protein and DNA polymerase to support viral genome replication and cccDNA synthesis. Not surprisingly, cytoplasmic expression of duck hepatitis B virus pgRNA initiated viral replication leading to infectious virion secretion. However, HBV replication and antiviral mechanism were studied primarily in human hepatoma cells transiently or stably transfected with plasmid-based HBV replicons. The presence of large amounts of transfected HBV DNA or transgenes in cellular chromosomes hampered the robust analyses of HBV replication and cccDNA function. As demonstrated here, the pgRNA launch HBV replication system permits the accurate mapping of antiviral target and investigation of cccDNA biosynthesis and transcription using secreted HBsAg as a convenient quantitative marker. The effect of drug-resistant variants on viral capsid assembly, genome replication, and cccDNA biosynthesis and function can also be assessed using this system.

Keywords: antiviral agents; capsid assembly; capsid disassembly; hepatitis B virus; viral replication.