Generation of Replication-Competent Hepatitis B Virus Harboring Tagged Polymerase for Visualization and Quantification of the Infection.

IF 1.9 4区医学 Q4 IMMUNOLOGY Microbiology and Immunology Pub Date : 2025-01-01 Epub Date: 2024-12-02 DOI:10.1111/1348-0421.13183

Chiharu Morita, Masami Wada, Eriko Ohsaki, Shihoko Kimura-Ohba, Keiji Ueda

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Abstract

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.

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携带标记聚合酶的复制型乙型肝炎病毒的产生用于感染的可视化和量化。

乙型肝炎病毒（HBV）感染是导致急性和慢性肝炎及相关疾病的严重全球卫生问题。大约有2.96亿患者慢性感染该病毒，导致肝硬化和肝细胞癌。虽然HBV聚合酶（HBVpol, pol）在HBV复制中起着关键作用，必须是一个明确的治疗靶点。问题是HBVpol的详细功能和细胞内动力学尚不清楚。在此，我们构建了两种带标记的HBVpol， PA-tagged和HiBiT-tagged pol，以及hbv产生载体。将每个PA标签和HiBiT标签插入HBVpol开放阅读框的间隔区n端。将质粒转染到HepG2细胞中导致HBV的产生。这些标记的HBVpol可以在HBV复制细胞中检测到，并且pol-HiBiT可以进行定量分析。此外，这些重组HBV对原代人肝细胞具有传染性。因此，我们成功地设计了具有传染性和复制能力的重组HBV，其中含有可检测的标记HBVpol。这种传染性重组HBV将为研究HBVpol动力学和开发新的HBV治疗方法提供新的工具。

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来源期刊

Microbiology and Immunology 医学-免疫学

CiteScore

5.20

自引率

3.80%

发文量

审稿时长

1 months

期刊介绍： Microbiology and Immunology is published in association with Japanese Society for Bacteriology, Japanese Society for Virology, and Japanese Society for Host Defense Research. It is peer-reviewed publication that provides insight into the study of microbes and the host immune, biological and physiological responses. Fields covered by Microbiology and Immunology include:Bacteriology|Virology|Immunology|pathogenic infections in human, animals and plants|pathogenicity and virulence factors such as microbial toxins and cell-surface components|factors involved in host defense, inflammation, development of vaccines|antimicrobial agents and drug resistance of microbes|genomics and proteomics.