Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
J Mol Biol. 2013 Mar 25;425(6):1050-64. doi: 10.1016/j.jmb.2013.01.004. Epub 2013 Jan 11.
We have examined the roles of RNA-coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises a β-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem-loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T=1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8-11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal-CP interactions.
我们研究了 RNA 外壳蛋白（CP）相互作用在卫星烟草坏死病毒（STNV）组装中的作用。病毒基因组 RNA 仅编码 CP，CP 由连接到带正电荷的 N 端延伸的 β 桶域组成。在该系统以前的晶体结构中，蛋白质的前 11 个残基无序。使用针对 CP 分离的 RNA 适体序列变体 B3，我们研究了 RNA 诱导组装的序列特异性。B3 由呈现四环序列 ACAA 的茎环组成。有一个明确的偏好，涵盖这个环序列的 RNA，如由 T = 1 衣壳的产量来衡量，这对茎内的序列是不敏感的。含有 B3 的病毒样颗粒已被结晶，并确定其结构为 2.3Å。还计算了涵盖 RNA 的较低分辨率图。B3 的存在导致位于颗粒 3 倍轴上的 N 端螺旋的有序性增加，这些螺旋延伸约一个半圈以包含残基 8-11，包括 R8 和 K9。在组装条件下，没有 RNA 的 STNV CP 是单体的，不会自我组装。这些事实表明，一个合理的组装起始模型是特定的 RNA 诱导稳定三聚体衣壳。螺旋延伸的基本性质表明，CP 之间的静电排斥阻止了在没有 RNA 的情况下的组装，并且这种障碍被正确定向的螺旋 RNA 茎的适当放置克服。这样的机制将与这里显示的用于组装较长 RNA 片段（包括 STNV 基因组）的结果一致。结果根据涉及由多个 RNA 包装信号-CP 相互作用驱动的基因组 RNA 压缩的组装的第一阶段进行讨论。
