HBV的核心蛋白异位调节因子不的改变cccDNA生物合成从新生

newchinabok

http://journals.plos.org/plospat ... ournal.ppat.1006658

antiHBVren

Abstract

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

Author summary

Persistent HBV infection relies on stable maintenance of a nuclear episomal viral genome called covalently closed circular (ccc) DNA, the sole transcriptional template supporting viral replication. The currently available antiviral therapeutics fail to cure chronic HBV infection due to their failure to eradicate or inactivate cccDNA. In addition to packaging viral pregenomic (pg) RNA and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, HBV core protein also participates in and regulates virion particle assembly, capsid uncoating and cccDNA formation. We report herein an intriguing observation that selected core protein allosteric modulators not only inhibit nucleocapsid assembly, but can also act on assembled, nucleus-bound nucleocapsids to promote their uncoating and consequentially interfere with cccDNA biosynthesis. This finding establishes molecular basis for development of novel core protein targeting antiviral agents with improved efficacy of suppressing cccDNA synthesis and curing chronic HBV infection.

摘要
乙型肝炎病毒（HBV）核心蛋白将病毒前基因组（pg）RNA和DNA聚合酶组装成核衣壳，以进行逆转录DNA复制。已发现若干种小分子化学型（包括杂芳基二氢嘧啶（HAPs）和氨磺酰苯甲酰胺（SBAs））变构调节核心蛋白质结构并随之改变核心蛋白质装配的动力学和途径，导致形成不规则形状的核心蛋白质聚集体或“空的“衣壳没有前基因组RNA和病毒DNA聚合酶。有趣的是，除了抑制核衣壳装配和随后的病毒基因组复制外，我们现在已经证明HAP和SBAs通过诱导从病毒颗粒衍生的核衣壳的分解从差异感染和细胞内扩增路径差异地调节共价闭合环状（ccc）DNA的生物合成以及在细胞质中含有双链DNA的子代核衣壳。具体而言，在核心壳体未涂布的时间不合适的情况下，可防止肝细胞从头感染期间的cccDNA形成，而瞬时加速来自细胞质子代核衣壳的cccDNA合成。我们的研究表明，正链DNA的延伸诱导核衣壳的结构变化，赋予成熟的核衣壳结合CpAMs并触发其解体的能力。了解核心蛋白质变构调节剂对核衣壳装配和拆卸的双重作用的分子机制将有助于发现新的核心蛋白靶向抗病毒剂，其可以更有效地抑制cccDNA合成并治愈慢性乙型肝炎。

作者总结
持续性HBV感染依赖于稳定维持核附加型病毒基因组称为共价闭合环（ccc）DNA，这是支持病毒复制的唯一转录模板。目前可用的抗病毒治疗剂由于未能根除或灭活cccDNA而不能治愈慢性HBV感染。 HBV核心蛋白除了将病毒前基因组（pg）RNA和DNA聚合酶复合物包装到核衣壳中以发生逆转录DNA复制外，还参与并调节病毒颗粒装配，衣壳脱壳和cccDNA形成。我们在这里报告一个有趣的观察，选择核心蛋白变构调节剂不仅抑制核衣壳装配，但也可以作用于装配，核绑定核衣壳，以促进其脱壳，并相应地干扰cccDNA生物合成。这一发现为开发靶向抗病毒药物的新型核心蛋白奠定了分子基础，具有抑制cccDNA合成和治疗慢性HBV感染的更好效果。