Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.
Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.
复制标题
RNA和外套蛋白基底的互构构象构象转换有效地组装病毒式衣壳。
DOI：
10.1016/j.jmb.2010.05.058
复制
发表时间：
2010-08-13
影响因子：
5.6
通讯作者：
Stockley PG
中科院分区：
文献类型：
--
作者：
Rolfsson Ó;Toropova K;Ranson NA;Stockley PG
单链RNA病毒将它们的基因组包装成具有固定体积的衣壳。我们分析了噬菌体MS 2外壳蛋白包装其基因组单链RNA的大的、确定的片段的能力。我们表明，包装成T=3衣壳在体外的效率是成反比的RNA长度，这意味着有一个自由能障碍，以克服在组装过程中。所有的RNA检查有更大的解决方案的持久性长度比他们成为包装的衣壳的内径，这表明蛋白质介导的RNA压缩必须发生在组装。将溴化乙锭结合到这些RNA片段中的一个上，这预计会降低其灵活性，严重抑制包装，与这一想法一致。在这些实验中用亚基因组RNA组装的衣壳的Cryo-EM结构显示在外壳蛋白壳下的RNA密度层，但缺乏在野生型病毒体中观察到的内部RNA壳的密度。当在组装反应中使用全长病毒体RNA时，内层被恢复，这意味着只有当衣壳被填充时，它才变得有序，这可能是因为空间和/或静电排斥的影响。冷冻-EM结果解释了封装的长度依赖性。此外，他们表明，对于亚基因组片段，最强的有序RNA密度出现在形成衣壳的二十面体5倍轴的外壳蛋白二聚体下方。在2倍轴处的蛋白质下方几乎没有这样的密度，这与我们的模型一致，其中外壳蛋白二聚体与位于整个基因组中的位点处的RNA茎环结合导致其优选构象的切换，从而调节构建T=3衣壳所需的准构象异构体的放置。这些数据与RNA和外壳蛋白构象的相互陪伴一致，部分解释了这些病毒如此快速和准确地组装的能力。
Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem–loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.