Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2.
Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2.
复制标题
DOI：
10.1016/j.jmb.2015.11.014
复制
发表时间：
2016-01-29
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;Middleton S;Manfield IW;White SJ;Fan B;Vaughan R;Ranson NA;Dykeman E;Twarock R;Ford J;Kao CC;Stockley PG
使用交联偶联基质辅助激光解吸/电离质谱和CLIP-Seq测序，我们确定了在衣壳蛋白和噬菌体MS 2的基因组RNA之间的界面处的肽和寡核苷酸序列。结果表明，相同的外壳蛋白（CP）-RNA和成熟蛋白（MP）-RNA接口中使用的每一个病毒颗粒。与CP亚基接触的病毒RNA部分跨越基因组，与每个颗粒内大量离散和相似的接触一致。许多这些网站相匹配的位置，多个，分散和简并的RNA位点与同源CP亲和力称为包装信号（PS）的先前的预测。化学RNA足迹法用于比较无蛋白基因组片段和病毒体中RNA的二级结构。一些PS部分存在于无蛋白质的RNA中，但其他PS需要从其主要溶液构象重折叠以形成病毒体中鉴定的接触。MP内的RNA结合肽映射到蛋白质N-末端一半的两个部分。比较MP序列从相关的cDNA表明一个类似的安排RNA结合位点，虽然这些N-末端区域只有有限的序列保守性。相反，C-末端的序列是高度保守的，与它们包含与宿主细胞初始接触所需的菌毛蛋白结合结构域一致。这些结果提供了独立的和明确的支持，通过PS介导的机制，涉及一系列的诱导适合病毒蛋白与RNA的相互作用的MS 2病毒粒子的组装。CLIP-Seq揭示了单链RNA病毒体中一组确定的RNA-外壳相互作用。RNA位点与先前对多个PS的预测相匹配。数据提供了明确的确认PS介导的组装。
Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)–RNA and maturation protein (MP)–RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA. CLIP-Seq reveals a defined set of RNA–coat interactions in a single-stranded RNA virion. RNA sites match previous predictions of multiple PSs. Data provide unambiguous confirmation of PS-mediated assembly.