Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus
Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus
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DOI：
10.1261/rna.1367209
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发表时间：
2009-03-01
期刊：
影响因子：
4.5
通讯作者：
Barr, John N.
中科院分区：
文献类型：
--
作者：
Mohl, Bjorn-Patrick;Barr, John N.
布尼亚韦拉病毒（Bunyamwera virus，BUNV）是负链RNA病毒的正布尼亚病毒属（Orthobunyavirus）和布尼亚病毒科（Bunyaviridae）的原型成员。与所有负链RNA病毒一样，BUNV基因组链和反基因组链不是裸RNA，而是沿着其整个长度与病毒编码的核衣壳（N）蛋白结合以形成核糖核蛋白（RNP）复合物。这种关联对于负链RNA病毒的生命周期至关重要，因为只有RNP对于生产性RNA合成和RNA包装具有活性。我们有兴趣了解N和RNA成分如何在布尼亚病毒RNP内结合的分子细节，以及是什么支配了病毒复制产物的明显选择性结合。为了实现这一目标，我们最近设计了一个协议，允许生成天然BUNV N蛋白，在生理条件下保持溶解度，并允许形成晶体，产生高分辨率的X射线衍射数据。在这里，我们扩展这项工作表明，这种可溶性N蛋白能够寡聚化和结合RNA，形成一个高度均匀的RNP复合物，表现出与病毒RNP共同的特征。通过提取和测序与这些模型RNP结合的RNA，我们确定N-RNA缔合的化学计量类似于每N单体12个核苷酸。此外，我们定义了BUNV RNA复制的最小序列要求。通过比较这个最小序列与我们的模型RNP结合的那些序列，我们得出结论，N蛋白并不强制性地需要一个序列或结构来进行RNA折叠。
Bunyamwera virus (BUNV) is the prototypic member of both the Orthobunyavirus genus and the Bunyaviridae family of negative stranded RNA viruses. In common with all negative stranded RNA viruses, the BUNV genomic and anti-genomic strands are not naked RNAs, but instead are encapsidated along their entire lengths with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex. This association is critical for the negative strand RNA virus life cycle because only RNPs are active for productive RNA synthesis and RNA packaging. We are interested in understanding the molecular details of how N and RNA components associate within the bunyavirus RNP, and what governs the apparently selective encapsidation of viral replication products. Toward this goal, we recently devised a protocol that allowed generation of native BUNV N protein that maintained solubility under physiological conditions and allowed formation of crystals that yielded high-resolution x-ray diffraction data. Here we extend this work to show that this soluble N protein is able to oligomerize and bind RNA to form a highly uniform RNP complex, which exhibits characteristics in common with the viral RNP. By extracting and sequencing RNAs bound to these model RNPs, we determined the stoichiometry of N-RNA association to be similar to 12 nucleotides per N monomer. In addition, we defined the minimal sequence requirement for BUNV RNA replication. By comparing this minimal sequence to those bound to our model RNP, we conclude that N protein does not obligatorily require a sequence or structure for RNA encapsidation.