Ludgate Laurie, Liu Kuancheng, Luckenbaugh Laurie, Streck Nicholas, Eng Stacey, Voitenleitner Christian, Delaney William E, Hu Jianming
Department of Microbiology and Immunology, Penn State University College of Medicine, Hershey, Pennsylvania, USA.
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
J Virol. 2016 May 27;90(12):5830-5844. doi: 10.1128/JVI.00394-16. Print 2016 Jun 15.
Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.
Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.
乙型肝炎病毒（HBV）核心蛋白（HBc）的多个亚基组装成一个二十面体衣壳，该衣壳包裹病毒前基因组RNA（pgRNA）。在高HBc和/或盐浓度条件下，即使没有pgRNA或任何其他病毒或宿主因子，HBc的N端结构域（NTD）也足以进行衣壳组装。C端结构域（CTD）被认为对于衣壳组装是可有可无的，尽管它对于pgRNA包装至关重要。我们在此报告，当在哺乳动物细胞裂解物、兔网织红细胞裂解物（RRL）中表达的HBc（低纳摩尔浓度）模拟体内病毒复制条件下达到的浓度，且远低于先前体外衣壳组装所用浓度时，能够组装成衣壳。此外，在RRL中生理低HBc浓度下，NTD不足以进行衣壳组装，CTD也是必需的。CTD可能通过RNA结合和蛋白质 - 蛋白质相互作用在这些条件下促进组装。此外，CTD在RRL中经历了与体内类似的磷酸化和去磷酸化事件，这些事件调节衣壳组装。重要的是，单独的NTD在哺乳动物细胞中也无法积累，可能是由于其未能有效组装。全长HBc的共表达挽救了RRL中的NTD组装以及哺乳动物细胞中的NTD表达和组装，导致形成同时包含全长HBc和NTD的镶嵌衣壳。这些结果对复制过程中的HBV组装具有重要意义，并提供了一个简便的无细胞系统来研究生理相关条件下的衣壳组装，包括宿主因子对其的调节。
乙型肝炎病毒（HBV）是一种重要的全球人类病原体，也是全球肝癌的主要病因。HBV的一个重要组成部分是由单一蛋白质核心蛋白（HBc）的多个拷贝组成的球形衣壳。我们开发了一种哺乳动物无细胞系统，其中HBc在生理（低）浓度下表达，并在接近生理条件下组装成衣壳。在这个无细胞系统中，与在哺乳动物细胞中一样，衣壳组装依赖于HBc的C端结构域（CTD），这与其他组装系统不同，在其他系统中，HBc在非生理蛋白质和盐浓度条件下独立于CTD组装成衣壳。此外，CTD的磷酸化状态以与在哺乳动物细胞中类似的方式调节无细胞系统中的衣壳组装和RNA包裹。该系统将有助于在生理条件下对衣壳组装和RNA包裹进行详细研究，并有助于鉴定靶向HBc的抗病毒药物。
