Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity
Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity
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DOI：
10.1128/jvi.00586-10
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发表时间：
2010-07-01
影响因子：
5.4
通讯作者：
Zlotnick, Adam
中科院分区：
文献类型：
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作者：
Porterfield, J. Zachary;Dhason, Mary Savari;Zlotnick, Adam
病毒生命周期的一个关键特征是有选择地包装病毒基因组的能力。在体内，磷酸化的乙型肝炎病毒（HBV）核心蛋白特异性地封装了基因组前RNA （pgRNA）和病毒聚合酶的复合物；有人建议，包装是特定的复合物。在这里，我们验证了核心蛋白对pgRNA具有内在特异性的假设，独立于聚合酶。在这些研究中，我们还评估了核心蛋白磷酸化对组装和RNA结合的影响，使用磷酸化的核心蛋白和磷酸化模拟物，其中S155， S162和S170突变为谷氨酸。我们已经开发了一种体外系统，其中衣壳被分解和组装活性核心蛋白二聚体被纯化。利用这种蛋白质，我们重新组装了空衣壳和充满rna的衣壳。我们发现，无论磷酸化与否，核心蛋白二聚体都能以高水平的协同性结合和封装HBV前基因组RNA和异源RNA。在直接竞争分析中，未观察到对前基因组RNA的特异性。这表明，另一个因素，如病毒聚合酶，需要特定的包装。这些结果也提出了一个问题，即是什么阻止了HBV核心蛋白在非病毒RNA上组装，从而为病毒的产生保留了蛋白质。
A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.