Morita Chiharu, Wada Masami, Ohsaki Eriko, Kimura-Ohba Shihoko, Ueda Keiji
Division of Virology, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Osaka, Japan.
Center for Infectious Disease Education and Research (CiDER), Osaka, Japan.
Microbiol Immunol. 2025 Jan;69(1):43-58. doi: 10.1111/1348-0421.13183. Epub 2024 Dec 2.
Hepatitis B virus (HBV) infection is a serious global health problem causing acute and chronic hepatitis and related diseases. Approximately, 296 million patients have been chronically infected with the virus, leading to cirrhosis and hepatocellular carcinoma. Although HBV polymerase (HBVpol, pol) plays a pivotal role in HBV replication and must be a definite therapeutic target. The problems are that the detailed functions and intracellular dynamics of HBVpol remain unclear. Here, we constructed two kinds of tagged HBVpol, PA-tagged and HiBiT-tagged pol, and the HBV-producing vectors. Each PA tag and HiBiT tag were inserted into N-terminus of spacer region on HBVpol open reading frame. Transfection of the plasmids into HepG2 cells led to production of HBV. These tagged HBVpol were detectable in HBV replicating cells and pol-HiBiT enabled quantitative analysis. Furthermore, these recombinant HBV were infectious to primary human hepatocytes. Thus, we successfully designed infectious and replication-competent recombinant HBV harboring detectable tagged HBVpol. Such infectious recombinant HBV will provide a novel tool to study HBVpol dynamics and develop new therapeutics against HBV.
乙型肝炎病毒（HBV）感染是一个严重的全球健康问题，可导致急性和慢性肝炎及相关疾病。大约有2.96亿患者长期感染该病毒，进而引发肝硬化和肝细胞癌。尽管HBV聚合酶（HBVpol，pol）在HBV复制中起关键作用，且必定是一个明确的治疗靶点。但问题在于，HBVpol的详细功能和细胞内动态仍不清楚。在此，我们构建了两种标记的HBVpol，即PA标记和HiBiT标记的pol，以及产生HBV的载体。每个PA标签和HiBiT标签都插入到HBVpol开放阅读框间隔区的N端。将这些质粒转染到HepG2细胞中可导致HBV的产生。这些标记的HBVpol在HBV复制细胞中可被检测到，且pol-HiBiT能够进行定量分析。此外,这些重组HBV对原代人肝细胞具有感染性。因此，我们成功设计出了带有可检测标记HBVpol的具有感染性和复制能力的重组HBV。这种感染性重组HBV将为研究HBVpol动态和开发抗HBV新疗法提供一种新工具。
