Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid.
Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid.
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DOI：
10.1016/j.jmb.2009.11.018
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发表时间：
2010-02-05
影响因子：
5.6
通讯作者：
Stockley PG
中科院分区：
文献类型：
--
作者：
Basnak G;Morton VL;Rolfsson O;Stonehouse NJ;Ashcroft AE;Stockley PG
单链RNA病毒控制基因组包装的分子机制在很大程度上仍然是未知的。在大多数情况下，为了由单个蛋白质的多个拷贝形成病毒衣壳，蛋白质在衣壳晶格的不同位置采用不同的构象是必要的。我们之前的研究表明，这种RNA噬菌体MS2外壳蛋白二聚体（CP2）的准等效构象可以通过与短RNA茎环（TR）的序列特异性相互作用来切换，这种相互作用在野生型噬菌体基因组中只发生一次。原则上，产生噬菌体T=3衣壳需要多个开关事件。因此，我们研究了这一事件的序列依赖性，使用了选择的两个RNA适体序列来结合噬菌体外壳蛋白和来自噬菌体Qβ的类似包装信号，已知这些信号在体内和体外都被MS2外壳蛋白歧视。所有三个非同源茎环都支持T=3壳的形成，但没有一个显示出当TR与等摩尔CP2混合时的动力学捕获效应。我们表明，这反映了这样一个事实，即与TR相比，它们是较差的配体，在实验条件下无法使外壳蛋白饱和，从而确保在这些反应中存在有效组装所需的两种类型的二聚体的足够数量。增加非同源RNA浓度恢复了动力学陷阱，证实了这一解释。我们还评估了用短基因组序列在5 ‘或3 ’端延长TR茎环的效果。这些较长的rna都显示出动力学陷阱的证据，反映了它们都含有TR序列，并且比TR更有效地促进衣壳形成的事实。质谱分析表明，在TR诱导的组装反应中，至少有两种途径通向T=3壳：一种是通过形成3倍轴，另一种是通过形成延伸的5倍复合物。较长的基因组rna抑制了5重通路，可能是多重结合rna之间的空间冲突的结果。与TR茎环相反的延伸序列方向产生的rna是不良的组装启动器。这些数据支持rna诱导的蛋白质构象转换发生在T=3壳的整个组装过程中，并表明TR茎环外的位置和序列特异性效应都可能对随后的精确组装途径产生重大影响。
The molecular mechanisms controlling genome packaging by single-stranded RNA viruses are still largely unknown. It is necessary in most cases for the protein to adopt different conformations at different positions on the capsid lattice in order to form a viral capsid from multiple copies of a single protein. We showed previously that such quasi-equivalent conformers of RNA bacteriophage MS2 coat protein dimers (CP2) can be switched by sequence-specific interaction with a short RNA stem–loop (TR) that occurs only once in the wild-type phage genome. In principle, multiple switching events are required to generate the phage T=3 capsid. We have therefore investigated the sequence dependency of this event using two RNA aptamer sequences selected to bind the phage coat protein and an analogous packaging signal from phage Qβ known to be discriminated against by MS2 coat protein both in vivo and in vitro. All three non-cognate stem–loops support T=3 shell formation, but none shows the kinetic-trapping effect seen when TR is mixed with equimolar CP2. We show that this reflects the fact that they are poor ligands compared with TR, failing to saturate the coat protein under the assay conditions, ensuring that sufficient amounts of both types of dimer required for efficient assembly are present in these reactions. Increasing the non-cognate RNA concentration restores the kinetic trap, confirming this interpretation. We have also assessed the effects of extending the TR stem–loop at the 5′ or 3′ end with short genomic sequences. These longer RNAs all show evidence of the kinetic trap, reflecting the fact that they all contain the TR sequence and are more efficient at promoting capsid formation than TR. Mass spectrometry has shown that at least two pathways toward the T=3 shell occur in TR-induced assembly reactions: one via formation of a 3-fold axis and another that creates an extended 5-fold complex. The longer genomic RNAs suppress the 5-fold pathway, presumably as a consequence of steric clashes between multiply bound RNAs. Reversing the orientation of the extension sequences with respect to the TR stem–loop produces RNAs that are poor assembly initiators. The data support the idea that RNA-induced protein conformer switching occurs throughout assembly of the T=3 shell and show that both positional and sequence-specific effects outside the TR stem–loop can have significant impacts on the precise assembly pathway followed.