THE ENCAPSIDATION SIGNAL ON THE HEPATITIS-B VIRUS-RNA PREGENOME FORMS A STEM-LOOP STRUCTURE THAT IS CRITICAL FOR ITS FUNCTION
THE ENCAPSIDATION SIGNAL ON THE HEPATITIS-B VIRUS-RNA PREGENOME FORMS A STEM-LOOP STRUCTURE THAT IS CRITICAL FOR ITS FUNCTION
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DOI：
10.1093/nar/21.17.3967
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发表时间：
1993-08-25
影响因子：
14.9
通讯作者：
NASSAL, M
中科院分区：
文献类型：
--
作者：
KNAUS, T;NASSAL, M
乙型肝炎病毒 (HBV) 是嗜肝DNA病毒科的类型成员，是一种小包膜 DNA 病毒，通过 RNA 中间体（前基因组）的逆转录进行复制。这种反应通常发生在病毒核衣壳内部，其组装需要核心蛋白、病毒复制酶（P 蛋白）和 RNA 前基因组（也充当这两种蛋白的 mRNA）的多个拷贝之间的特定相互作用。删除研究已经证实，RNA 的特异性包装是由短顺式作用序列（衣壳化信号 epsilon）介导的。通过核酸酶敏感性实验，我们提供了实验证据，证明该序列的一部分可以采用被凸起和单个不配对的 U 残基打断的茎环结构。在体外研究了未配对区域的删除及其一级序列变化的结构后果，并通过监测在 2.7 kb 外来 RNA 片段前面或在完整 HBV 基因组范围内携带突变型 ε 序列的 RNA 的衣壳化，在动物细胞中测试了它们对 ε 信号功能的影响。数据表明，包含凸起和环的整个茎环结构对于衣壳封装能力至关重要。虽然未配对区域的一级序列的总体改变干扰衣壳化，但用其他突变体获得的数据表明凸出区域比环更能耐受序列变化。
Hepatitis B virus (HBV) is the type member of the hepadnaviridae, small enveloped DNA viruses that replicate through reverse transcription of an RNA intermediate, the pregenome. This reaction occurs usually inside the viral nucleocapsid, the assembly of which requires specific interactions between multiple copies of the core protein, the viral replication enzyme (P protein) and the RNA pregenome which also serves as mRNA for both proteins. Deletion studies have established that specific packaging of the RNA is mediated by a short cis-acting sequence, the encapsidation signal epsilon. Using nuclease sensitivity experiments we provide experimental evidence that part of this sequence can adopt a stem-loop structure that is interrupted by a bulge and a single unpaired U residue. The structural consequences of deletions of the unpaired regions and changes in their primary sequences were investigated in vitro, and their influence on the function of the epsilon-signal was tested in animal cells by monitoring encapsidation of RNAs carrying the mutant epsilon-sequences in front of a 2.7 kb foreign RNA fragment, or within the context of a complete HBV genome. The data indicate that the entire stem-loop structure containing the bulge and the loop is critical for encapsidation competence. While gross alterations in the primary sequences of the unpaired regions interfere with encapsidation, data obtained with additional mutants suggest that the bulge region is more tolerant to sequence changes than the loop.