Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.
Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts.
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DOI：
10.1371/journal.ppat.1009146
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发表时间：
2020-12
期刊：
影响因子：
6.7
通讯作者：
Stockley PG
中科院分区：
文献类型：
--
作者：
Chandler-Bostock R;Mata CP;Bingham RJ;Dykeman EC;Meng B;Tuthill TJ;Rowlands DJ;Ranson NA;Twarock R;Stockley PG
小核糖核酸病毒是重要的病毒病原体，但是尽管进行了广泛的研究，但其感染性病毒体的组装过程仍然不完全清楚，这阻碍了针对生命周期中这一重要部分的抗病毒策略的发展。我们报告的识别，通过RNA SELEX和生物信息学，在一个典型的肠道病毒，肠道病毒-E（EV-E），每个具有亲和力的同源病毒衣壳蛋白（CP）衣壳蛋白的基因组中的多个RNA位点。这些位点中的许多在已知的EV-E变体中是进化上保守的，这表明它们发挥着重要的功能作用。使用冷冻电子显微镜以~2.2 μ m分辨率重建EV-E颗粒，揭示基因组RNA的广泛密度。放松重建颗粒内强加的对称性揭示了多个RNA-CP接触，这是任何小核糖核酸病毒的第一次。EV-E中单个RNA接触氨基酸侧链的保守诱变是致命的，但不干扰复制或翻译，其中许多氨基酸侧链在包括脊髓灰质炎病毒在内的肠道病毒家族中是保守的。抗EV-E和抗脊髓灰质炎病毒适体与分布在脊髓灰质炎病毒基因组中的位点具有序列相似性。这些数据与以下假设一致，即这些RNA-CP接触是在组装中起重要作用的RNA包装信号（PS），并表明RNA PS在家族内的病原体之间进化上保守，增强了该病毒家族当前仅蛋白质组装的范例。小核糖核酸病毒是重要的病原体，但它们的组装不完全了解，阻碍了抗病毒药物和疫苗的开发。我们报告了多个RNA茎环的鉴定，每个环中有嘌呤三核苷酸序列基序，分布在两种肠道病毒，肠道病毒-E和脊髓灰质炎病毒的基因组中。每个基序似乎对其同源衣壳蛋白具有亲和力。没有强加对称性的肠道病毒-E的Cryo-EM重建鉴定出许多重复的CP-RNA接触。这些包括与颗粒二重轴相邻的茎环，其在病毒之间似乎是保守的，与所有基因组RNA片段接触的氨基酸侧链也是如此。突变表明，这些氨基酸是病毒体形成所必需的，与RNA依赖性组装机制一致。这些结果表明，所有的肠道病毒利用一个共同的装配机制，需要定义的CP-RNA接触。
Picornaviruses are important viral pathogens, but despite extensive study, the assembly process of their infectious virions is still incompletely understood, preventing the development of anti-viral strategies targeting this essential part of the life cycle. We report the identification, via RNA SELEX and bioinformatics, of multiple RNA sites across the genome of a typical enterovirus, enterovirus-E (EV-E), that each have affinity for the cognate viral capsid protein (CP) capsomer. Many of these sites are evolutionarily conserved across known EV-E variants, suggesting they play essential functional roles. Cryo-electron microscopy was used to reconstruct the EV-E particle at ~2.2 Å resolution, revealing extensive density for the genomic RNA. Relaxing the imposed symmetry within the reconstructed particles reveals multiple RNA-CP contacts, a first for any picornavirus. Conservative mutagenesis of the individual RNA-contacting amino acid side chains in EV-E, many of which are conserved across the enterovirus family including poliovirus, is lethal but does not interfere with replication or translation. Anti-EV-E and anti-poliovirus aptamers share sequence similarities with sites distributed across the poliovirus genome. These data are consistent with the hypothesis that these RNA-CP contacts are RNA Packaging Signals (PSs) that play vital roles in assembly and suggest that the RNA PSs are evolutionarily conserved between pathogens within the family, augmenting the current protein-only assembly paradigm for this family of viruses. Picornaviruses are important pathogens but their assembly is incompletely understood, preventing development of anti-viral drugs and vaccines. We report identification of multiple RNA stem–loops, with purine trinucleotide sequence motifs in each loop, distributed across the genomes of two enteroviruses, enterovirus-E and poliovirus. Each motif appears to have affinity for its cognate capsid protein. Cryo-EM reconstruction of enterovirus-E without imposed symmetry identifies many repeated CP-RNA contacts. These include stem-loops adjacent to the particle two-fold axes, which appear conserved between viruses, as are the amino acid side-chains that contact all the genomic RNA segments. Mutagenesis shows that these amino acids are essential for virion formation, consistent with an RNA-dependent assembly mechanism. These results suggest that all enteroviruses utilise a common assembly mechanism that requires defined CP-RNA contacts.