乙型肝炎病毒核心蛋白磷酸化：鉴定SRPK1靶位点及其占据对RNA

StephenW

PLoS Pathog. 2018 Dec 19;14(12):e1007488. doi: 10.1371/journal.ppat.1007488. [Epub ahead of print]
Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure.
Heger-Stevic J1,2, Zimmermann P1,2, Lecoq L3, Böttcher B4, Nassal M1.
Author information

1
    University Hospital Freiburg, Department of Medicine II / Molecular Biology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
2
    Biological Faculty, University of Freiburg, Freiburg, Germany.
3
    Institut de Biologie et Chimie des Protéines, University of Lyon1, Lyon, France.
4
    Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany.

Abstract

Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.

PMID:
    30566530
DOI:
    10.1371/journal.ppat.1007488

StephenW

PLoS Pathog。 2018年12月19日; 14（12）：e1007488。 doi：10.1371 / journal.ppat.1007488。 [提前打印]
乙型肝炎病毒核心蛋白磷酸化：鉴定SRPK1靶位点及其占据对RNA结合和衣壳结构的影响。
Heger-Stevic J1,2，Zimmermann P1,2，Lecoq L3，BöttcherB4，Nassal M1。
作者信息

1
    弗莱堡大学医院，医学系II /分子生物学，德国弗莱堡弗莱堡大学医学院。
2
    德国弗莱堡弗莱堡大学生物系。
3
    法国里昂里昂大学生物学与生物学研究所。
4
    德国维尔茨堡维尔茨堡大学生物中心生物化学系。

抽象

乙型肝炎病毒（HBV）通过衣壳内部逆转录复制其3kb DNA基因组，通过在病毒前基因组（pg）RNA和聚合酶的复合物周围组装120个核心蛋白（HBc）二聚体启动。合成松弛的环状（RC）DNA衣壳后，可以包裹并分泌稳定的病毒粒子。然而，在感染新细胞后，衣壳解体以将RC-DNA释放到细胞核中以转化成共价闭合的环状（ccc）DNA。 HBc与核酸的相互作用由富含精氨酸的C末端结构域（CTD）介导，具有固有强的非特异性RNA结合活性。在病毒生命周期期间适应对核酸结合的变化需求被认为涉及动态磷酸化/去磷酸化事件。然而，HBc中的相关酶和它们的靶位点都没有牢固确立。在这里，我们开发了一种细菌共表达系统，可以获得可定义的磷酸化HBc。结合Phos标签凝胶电泳，质谱和诱变，我们发现CTD中的8个羟基氨基酸中的7个作为富含丝氨酸 - 精氨酸的蛋白激酶1（SRPK1）的靶位点; PKA和PKC磷酸化的位点较少。所有七个位点的磷酸化均大大减少了非特异性RNA衣壳化，如同整个CTD的缺失和改变的CTD表面可及性，而衣壳壳中没有主要的结构变化。来自人肝癌细胞的大量衣壳与SRPK1一样高度但不同地磷酸化。虽然没有证明SRPK1是感染相关的HBc激酶，但数据表明高水平HBc磷酸化主要抑制RNA结合的机制，而一个或几个战略性去磷酸化事件能够选择性包装pgRNA /聚合酶复合物。本研究中开发的工具应极大地促进进一步破译HBc磷酸化在HBV感染中的作用及其作为潜在新治疗靶点的评价。

结论：
    30566530
DOI：
    10.1371 / journal.ppat.1007488

StephenW

https://journals.plos.org/plospa ... ournal.ppat.1007488