乙型肝炎病毒核心蛋白去磷酸化在前基因组RNA封装期间发生

StephenW

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation

    Qiong Zhaoa, Zhanying Hua, Junjun Chenga, Shuo Wua, Yue Luoa,b, Jinhong Changa, Jianming Huc and Ju-Tao Guoa

    aBaruch S. Blumberg Institute, Doylestown, Pennsylvania, USA
    bInstitute of Hepatology, Second Xiangya Hospital, Central South University, Changsha, Hunan Province, China
    cDepartment of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA

    J.-H. James Ou, Editor

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Author Affiliations

    University of Southern California

ABSTRACT

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically “normal” capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.

IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.

StephenW

乙型肝炎病毒核心蛋白去磷酸化在前基因组RNA封装期间发生

    琼兆娅，占英华，君俊成，硕沃亚，罗罗亚，金宏昌，胡建明，郭巨涛

    aBaruch S. Blumberg研究所，美国宾夕法尼亚州Doylestown
    b中南大学湘雅二医院肝病研究所，湖南长沙，中国
    美国宾夕法尼亚州赫尔希宾夕法尼亚州立大学医学院微生物与免疫学系

    J.-H. James Ou，编辑

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作者附属机构

    南加州大学

抽象

乙型肝炎病毒（HBV）核心蛋白由N-末端装配结构域和C-末端结构域（CTD）组成，其具有7个保守的丝氨酸或苏氨酸，其在病毒复制周期期间被动态磷酸化/去磷酸化。氨磺酰苯甲酰胺衍生物是与核心蛋白二聚体 - 二聚体界面之间的杂芳基二氢嘧啶（HAP）口袋结合的小分子核心蛋白质变构调节剂（CpAMs）。 CpAM结合改变衣壳装配的动力学和途径，并可导致形成没有病毒前基因组RNA（pgRNA）和DNA聚合酶的形态学“正常”衣壳。为了研究CpAM抑制pgRNA衣壳化的机制，我们开发了可以根据其磷酸化状态解析核心蛋白的免疫印迹测定，并且首次证明核心蛋白在游离二聚体中是超磷酸化的并且来自两种模拟的空壳体处理和CpAM处理的细胞，但在含pgRNA和DNA的核衣壳中低磷酸化。有趣的是，热休克蛋白90（HSP90）抑制剂抑制pgRNA衣壳化可防止核心蛋白去磷酸化。此外，在HAP口袋壁上具有点突变的核心蛋白V124A和V124W组装了具有改变的磷酸化状态的空壳体和核衣壳。结果表明核心蛋白去磷酸化发生在pgRNA的组装中，并且核衣壳装配过程中干扰二聚体 - 二聚体界面处的核心蛋白亚基之间的相互作用不仅改变衣壳结构，而且改变核心蛋白去磷酸化。因此，通过CpAM抑制pgRNA衣壳化可能是由于核衣壳装配过程中核心蛋白去磷酸化的破坏。

重要性HBV核心蛋白的动态磷酸化调节病毒复制的多个步骤。然而，调节功能主要是通过磷酸模拟突变来研究的，磷酸模拟突变破坏了核心蛋白磷酸化/去磷酸化的自然动态。开发一种能够分解高和低磷酸化核心蛋白的免疫印迹试验，使我们能够追踪作为游离二聚体存在的核心蛋白和细胞内衣壳的多样性的磷酸化状态，并研究核心蛋白磷酸化/去磷酸化在病毒复制中的作用。在这里，我们发现核衣壳装配期间（通过CpAM或诱变）在二聚体 - 二聚体界面处核心蛋白相互作用的破坏抑制了核心蛋白去磷酸化和pgRNA包装。因此，我们的工作揭示了核心蛋白去磷酸化在HBV复制中的新功能，以及目前正在临床试验中用于治疗慢性乙型肝炎的一类化合物CpAMs诱导空衣壳装配的机制。