Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome.

IF 3.7 2区 生物学 Q2 MICROBIOLOGY mSphere Pub Date : 2024-10-29 Epub Date: 2024-09-30 DOI:10.1128/msphere.00518-24
Asako Murayama, Hitomi Igarashi, Norie Yamada, Hussein Hassan Aly, Masaaki Toyama, Masanori Isogawa, Tetsuro Shimakami, Takanobu Kato
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Abstract

A cell culture system that allows the reproduction of the hepatitis B virus (HBV) life cycle is indispensable to exploring novel anti-HBV agents. To establish the screening system for anti-HBV agents, we exploited the high affinity and bright luminescence (HiBiT) tag and comprehensively explored the regions in the HBV genome where the HiBiT tag could be inserted. The plasmids for the HiBiT-tagged HBV molecular clones with a 1.38-fold HBV genome length were prepared. The HiBiT tag was inserted into five regions: preS1, preS2, hepatitis B e (HBe), hepatitis B X (HBx), and hepatitis B polymerase (HB pol). HiBiT-tagged HBVs were obtained by transfecting the prepared plasmids into sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells, and their infectivity was evaluated in human primary hepatocytes and HepG2/NTCP cells. Among the evaluated viruses, the infection of HiBiT-tagged HBVs in the preS1 or the HB pol regions exhibited a time-dependent increase of the hepatitis B surface antigen (HBsAg) level after infection to HepG2/NTCP cells as well as human primary hepatocytes. Immunostaining of the hepatitis B core (HBc) antigen in infected cells confirmed these viruses are infectious to those cells. However, the time-dependent increase of the HiBiT signal was only detected after infection with the HiBiT-tagged HBV in the preS1 region. The inhibition of this HiBiT-tagged HBV infection in human primary hepatocytes and HepG2/NTCP cells by the preS1 peptide could be detected by measuring the HiBiT signal. The infection system with the HiBiT-tagged HBV in HepG2/NTCP cells facilitates easy, sensitive, and high-throughput screening of anti-HBV agents and will be a useful tool for assessing the viral life cycle and exploring antiviral agents.

Importance: Hepatitis B virus (HBV) is the principal causative agent of chronic hepatitis. Despite the availability of vaccines in many countries, HBV infection has spread worldwide and caused chronic infection. In chronic hepatitis B patients, liver inflammation leads to cirrhosis, and the accumulation of viral genome integration into host chromosomes leads to the development of hepatocellular carcinoma. The currently available treatment strategy cannot expect the eradication of HBV. To explore novel anti-HBV agents, a cell culture system that can detect HBV infection easily is indispensable. In this study, we examined the regions in the HBV genome where the high affinity and bright luminescence (HiBiT) tag could be inserted and established an HBV infection system to monitor infection by measuring the HiBiT signal by infecting the HiBiT-tagged HBV in sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. This system can contribute to screening for novel anti-HBV agents.

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探索乙型肝炎病毒基因组中 HiBiT 标记插入的可容忍区域。
要探索新型抗乙型肝炎病毒(HBV)制剂,就必须建立一个能够复制乙型肝炎病毒(HBV)生命周期的细胞培养系统。为了建立抗 HBV 药物的筛选系统,我们利用了高亲和力和明亮发光(HiBiT)标签,并全面探索了 HBV 基因组中可插入 HiBiT 标签的区域。我们制备了 HBV 基因组长度为 1.38 倍的 HiBiT 标记 HBV 分子克隆质粒。HiBiT 标记被插入五个区域:preS1、preS2、乙肝 e(HBe)、乙肝 X(HBx)和乙肝聚合酶(HB pol)。通过将制备好的质粒转染到牛磺胆酸钠共转运多肽转导的 HepG2(HepG2/NTCP)细胞中,获得了 HiBiT 标记的 HBV,并在人类原代肝细胞和 HepG2/NTCP 细胞中评估了它们的感染性。在评估的病毒中,前S1区或HB pol区的HiBiT标记的HBV感染HepG2/NTCP细胞和人类原代肝细胞后,乙肝表面抗原(HBsAg)水平的升高与时间有关。感染细胞中乙肝核心抗原(HBc)的免疫染色证实了这些病毒对这些细胞具有传染性。然而,只有在感染前 S1 区的 HiBiT 标记 HBV 后,才能检测到 HiBiT 信号随时间的增加。通过测量 HiBiT 信号,可以检测到 preS1 肽对人类原代肝细胞和 HepG2/NTCP 细胞中 HiBiT 标记 HBV 感染的抑制作用。HiBiT标记的HBV在HepG2/NTCP细胞中的感染系统有助于简便、灵敏和高通量地筛选抗HBV药物,并将成为评估病毒生命周期和探索抗病毒药物的有用工具:乙型肝炎病毒(HBV)是慢性肝炎的主要病原体。尽管许多国家都有疫苗,但乙型肝炎病毒感染已在全球蔓延并造成慢性感染。慢性乙型肝炎患者的肝脏炎症会导致肝硬化,病毒基因组整合到宿主染色体上的积累会导致肝细胞癌的发生。目前现有的治疗策略无法指望根除 HBV。要探索新型抗 HBV 药物,就必须建立一个能轻松检测 HBV 感染的细胞培养系统。在这项研究中,我们研究了 HBV 基因组中可插入高亲和力和明亮发光(HiBiT)标签的区域,并建立了一个 HBV 感染系统,通过在牛磺胆酸钠共转运多肽转导的 HepG2(HepG2/NTCP)细胞中感染 HiBiT 标签的 HBV,测量 HiBiT 信号来监测感染情况。该系统有助于筛选新型抗 HBV 药物。
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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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Shining a light on Candida-induced epithelial damage with a luciferase reporter. Strain variation in Candida albicans glycolytic gene regulation. The putative type 4 secretion system effector BspD is involved in maintaining envelope integrity of the pathogen Brucella. Burkholderia pseudomallei BopE suppresses the Rab32-dependent defense pathway to promote its intracellular replication and virulence. Chlamydia trachomatis Inc Ct226 is vital for FLI1 and LRRF1 recruitment to the chlamydial inclusion.
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