Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped
Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped
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DOI：
10.1099/0022-1317-80-10-2647
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发表时间：
1999-10-01
影响因子：
3.8
通讯作者：
Lo, SCJ
中科院分区：
文献类型：
--
作者：
Hui, EKW;Yi, YS;Lo, SCJ
乙型肝炎病毒 (HBV) 核衣壳的结构已通过冷冻电子显微镜详细揭示。核衣壳如何与表面抗原相互作用形成有包膜的病毒粒子仍然未知。在本研究中，创建具有 N 末端添加的核心突变体是为了解决两个问题：（1）这些突变体核心蛋白是否仍能形成核衣壳；（2）如果是这样，突变体核衣壳能否与表面抗原相互作用形成病毒粒子样颗粒。编码一段额外的 23 个氨基酸（包括 6 个组氨酸残基）的质粒与核心蛋白（称为 HisC 183）的 N 末端融合，在大肠杆菌中表达，并通过蛋白质印迹检测。 CsCl 梯度和电子显微镜分析表明 HisC183 可以自组装成核衣壳。当HisC183或另一种类似的N端融合核心蛋白（指定为FlagC183）与核心阴性质粒在人肝癌细胞中共表达时，两种突变核心蛋白均自组装成核衣壳，这些颗粒也保留了激酶活性。使用内源聚合酶测定，从突变核心形成的细胞内核衣壳中获得用同位素标记的填充型 HBV DNA。相反，从转染培养基中未检测到此类信号，这与 PCR 和 Southern blot 分析一致。结果表明，具有N端延伸的核心突变体可以形成核衣壳，但在包封过程中被阻断，并且不能形成分泌的病毒粒子。这项工作产生的突变核衣壳应该有助于进一步研究核衣壳如何与表面抗原相互作用。
The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC 183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids, These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.