Rewriting nature's assembly manual for a ssRNA virus.
Rewriting nature's assembly manual for a ssRNA virus.
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DOI：
10.1073/pnas.1706951114
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发表时间：
2017-11-14
影响因子：
11.1
通讯作者：
Stockley PG
中科院分区：
文献类型：
--
作者：
Patel N;Wroblewski E;Leonov G;Phillips SEV;Tuma R;Twarock R;Stockley PG
由外壳蛋白包裹ssRNA基因组组成的病毒是最简单的生物实体之一。它们的生命周期包括一系列过程，例如特定的基因组组装和有效的衣壳自组装。直到最近，这些都没有联系，但我们已经表明，许多病毒在这一类编码多个，简并RNA序列/结构基序结合同源外壳蛋白集体。这同时确保了特定的基因组包装和有效的病毒粒子组装通过RNA编码的指导手册。在这里，我们提取的病毒RNA基因组中的这本手册的基本特征，创建一个合成序列与组装基板优于天然等价物上级。此类RNA具有有效生产稳定的病毒样颗粒疫苗和/或基因/药物递送载体的潜力。卫星烟草坏死病毒（STNV）是已知的最小的病毒之一。它的基因组仅编码其外壳蛋白（CP）亚基，依靠其辅助病毒TNV的聚合酶进行复制。基因组已被证明含有一组神秘的分散的组装信号的茎环的形式，每个目前最小的CP结合基序AXXA的环。预测包含核苷酸1-127的基因组片段含有五个这样的包装信号（PS）。我们已经使用诱变来确定该区域中的关键组装特征。这些包括CP结合基序，PS茎环的相对位置，它们的数量和它们的折叠倾向。CP结合具有静电贡献，但组装成核由RNA片段中折叠PS的识别主导。去除整个基因组中PS中所有AXXA基序的突变产生不能有效组装的RNA。相比之下，当包含改进的PS的合成的127-nt片段被交换到否则缺乏CP识别基序的RNA上时，组装被部分恢复，尽管产生的病毒样颗粒是不完整的，这意味着该区域外的PS是正确组装所需的。将这个改进的区域交换到野生型STNV 1序列中会产生比病毒RNA更好的组装底物，产生完整的衣壳并在头对头竞争中击败野生型基因组。这些数据证实了PS介导的STNV组装机制的细节，并确定了一种有效的方法，用于生产稳定的病毒样颗粒包裹非天然RNA或其他货物。
Viruses composed of a shell of coat proteins enclosing ssRNA genomes are among the simplest biological entities. Their lifecycles include a range of processes, such as specific genome encapsidation and efficient capsid self-assembly. Until recently, these were not linked, but we have shown that many viruses in this class encode multiple, degenerate RNA sequence/structure motifs that bind cognate coat proteins collectively. This simultaneously ensures specific genome packaging and efficient virion assembly via an RNA-encoded instruction manual. Here we extract essential features of this manual in a viral RNA genome, creating a synthetic sequence with an assembly substrate superior to the natural equivalent. Such RNAs have the potential for efficient production of stable virus-like particle vaccines and/or gene/drug delivery vehicles. Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1–127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes.
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影响因子：
5.6
作者：
Ford, Robert J.;Barker, Amy M.;Bakker, Saskia E.;Coutts, Robert H.;Ranson, Neil A.;Phillips, Simon E. V.;Pearson, Arwen R.;Stockley, Peter G.
通讯作者：
Stockley, Peter G.
影响因子：
28.3
作者：
Patel N;White SJ;Thompson RF;Bingham R;Weiß EU;Maskell DP;Zlotnick A;Dykeman E;Tuma R;Twarock R;Ranson NA;Stockley PG
通讯作者：
Stockley PG
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。
影响因子：
5.6
作者：
Rolfsson Ó;Toropova K;Ranson NA;Stockley PG
通讯作者：
Stockley PG
影响因子：
5.6
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
通讯作者：
Stockley PG
影响因子：
14.9
作者：
Ding, Y;Chan, CY;Lawrence, CE
通讯作者：
Lawrence, CE