乙型肝炎病毒核心蛋白磷酸化：SRPK1靶位点的鉴定及其占据对

StephenW

Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

    Julia Heger-Stevic,
    Peter Zimmermann,
    Lauriane Lecoq,
    Bettina Böttcher,
    Michael Nassal

PLOS x

    Published: December 19, 2018
    https://doi.org/10.1371/journal.ppat.1007488



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.
Author summary

The liver-pathogenic hepatitis B virus (HBV) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic (pg)RNA. This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein (HBc) subunits. Capsid-internal formation of relaxed circular (RC) DNA enables the particle to leave the cell as stable virion; yet, when infecting a new cell it must release the RC-DNA for conversion into another, plasmid-like DNA that templates new viral RNAs. This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc, mainly in its arginine-rich C terminal domain (CTD) which displays strong non-sequence-specific RNA binding. However, neither the phosphorylation sites nor the relevant enzymes are well defined. We developed a recombinant system to produce kinase-specific phospho-HBc species, and adapted a feasible gel assay for their separation. By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase, SRPK1, in the CTD. As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved. Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target.

StephenW

乙型肝炎病毒核心蛋白磷酸化：SRPK1靶位点的鉴定及其占据对RNA结合和衣壳结构的影响

    Julia Heger-Stevic，
    彼得齐默尔曼，
    Lauriane Lecoq，
    BettinaBöttcher，
    迈克尔纳萨尔

PLOS x

    发布时间：2018年12月19日
    https://doi.org/10.1371/journal.ppat.1007488



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。
作者摘要

肝脏致病性乙型肝炎病毒（HBV）是一种小包膜DNA病毒，通过前基因组（pg）RNA的逆转录复制。这需要将pgRNA和病毒聚合酶特异性衣壳化成240个核心蛋白（HBc）亚基的壳。松弛环状（RC）DNA的衣壳内部形成使颗粒能够作为稳定的病毒颗粒离开细胞;然而，当感染新细胞时，它必须释放RC-DNA，转化为另一种质粒样DNA，模拟新的病毒RNA。这种核酸相互作用的上下推测可能是通过HBc的瞬时磷酸化来调节的，主要是在其富含精氨酸的C末端结构域（CTD）中，其显示出强烈的非序列特异性RNA结合。然而，磷酸化位点和相关酶都没有明确定义。我们开发了一种重组系统来生产激酶特异性磷酸化HBc物种，并采用可行的凝胶测定法进行分离。通过诱变和质谱分析，我们在CTD中鉴定了主要候选激酶SRPK1的7个靶位点。由于完全SRPK1磷酸化阻碍了非特异性RNA结合，人类细胞中HBc的相对高磷酸化表明可能如何实现特异性pgRNA衣壳化。我们的新工具集将有助于解开HBc磷酸化在HBV感染中的作用，并将其作为潜在的治疗靶点。

StephenW

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