Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells
Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells
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乙型肝炎病毒胞质核衣壳的有限分解限制了病毒在小鼠肝细胞中的感染
DOI：
10.1002/hep.32622
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发表时间：
2022-06
期刊：
影响因子：
13.5
通讯作者：
Kaitao Zhao;Fangteng Guo;Jingjing Wang;Youquan Zhong;J. Yi;Yan Teng;Zaichao Xu;Li Zhao;Aixin Li;Zichen Wang;Xinwen Chen;Xiaoming Cheng;Yuchen Xia
中科院分区：
文献类型：
--
作者：
Kaitao Zhao;Fangteng Guo;Jingjing Wang;Youquan Zhong;J. Yi;Yan Teng;Zaichao Xu;Li Zhao;Aixin Li;Zichen Wang;Xinwen Chen;Xiaoming Cheng;Yuchen Xia
背景与目的：小鼠肝细胞即使辅以病毒受体--人牛磺胆酸钠共转运多肽(HNTCP)的表达，仍不能支持乙肝病毒的感染。然而，具体的限制性步骤仍然难以捉摸。本研究旨在对小鼠肝细胞中乙肝病毒的感染过程进行研究。方法与结果：用乙肝病毒或丁型肝炎病毒(HDV)接种hNTCP表达细胞。在体外产生HBV前基因组RNA(PgRNA)、共价闭合环状DNA(CccDNA)和不同的松弛环状DNA(RcDNA)中间体。通过体外修复实验和小鼠流体力学注射修复实验，研究了rcDNA到cccDNA的修复过程。用Southern印迹和原位杂交检测HBVDNA。在表达hNTCP的小鼠肝细胞中，限制了HBV病毒，而不是其卫星病毒HDV的生产性感染。HBVpgRNA可以在人肝细胞中复制，但不能在小鼠肝细胞中复制。在小鼠肝细胞中，HBVcccDNA和重组ccccDNA可支持乙肝病毒的转录。不同的rcDNA中间体在体外和体内均可被修复形成cccDNA。此外，在小鼠肝细胞核中可检测到rcDNA，但不能形成ccDNA。有趣的是，核酸酶敏感性分析表明，从细胞质中分离到的蛋白连接的rcDNA在小鼠肝细胞中完全抗核酸酶，而在人肝细胞中不抗核酸酶。结论：我们的结果表明，在小鼠肝细胞中，胞浆内的乙肝病毒核衣壳的拆解受到限制。克服这一局限可能有助于建立一种乙肝病毒感染的小鼠模型。
Background and Aims: Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. Approach and Results: Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro. The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication‐competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo. In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein‐linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. Conclusions: Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.