PDF(Pubmed)
Abstract：
Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.
摘要：
丁型肝炎病毒（HDV）感染代表了慢性病毒性肝炎的最严重形式。我们已经表明，使用腺相关病毒（AAV-HDV）作为基因递送载体将HDV复制能力基因组递送到肝细胞提供了一个独特的平台来研究HDV和相关肝损伤的分子方面。出于本研究的目的，我们产生了通过定点诱变修饰的HDV基因组，旨在（i）防止HDV抗原（HDAg）的某些翻译后修饰，例如大HDAg（L-HDAg）异戊二烯化或短HDAg（S-HDAg）磷酸化;（ii）改变HDAg在亚细胞区室中的定位；（iii）抑制δ核酶的正确构象。首先,使用质粒转染的Huh-7细胞在体外测试不同的HDV突变体,然后使用AAV载体在C57BL/6小鼠中进行体内测试。我们发现Ser177磷酸化和核酶活性对于HDV复制和HDAg表达至关重要。异戊二烯化结构域的突变阻止了感染性颗粒的形成和增加的细胞毒性和肝损伤。此外，改变HDAg细胞内定位显着减少病毒复制，尽管肝损伤与正常HDAg分布相比保持不变。此外,核输出信号的突变损害了感染性病毒颗粒的形成。这些发现为HDV生物学的复杂机制提供了有价值的见解，并对治疗考虑具有意义。
