Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, UK.
Helmholtz Institute for RNA-based Infection Research (HIRI), Würzburg, Germany.
Nucleic Acids Res. 2024 Aug 12;52(14):8500-8514. doi: 10.1093/nar/gkae404.
How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA-RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation ('anchor') for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.
分段 RNA 病毒如何正确募集其基因组尚未阐明。蓝舌病毒是一种双链 RNA 病毒，具有 10 个大小不同的片段，但它通过一系列特定的 RNA-RNA 相互作用，在包装之前以单链形式组装其基因组。在这项研究中，我们使用 2'-羟基酰化分析引物延伸和突变分析（SHAPE-MaP）来确定每个 BTV 转录本的结构，分别和不同组合。SHAPE-MaP 确定了在复合物形成过程中和可能的 RNA-RNA 相互作用位点中的 RNA 结构变化。我们的数据还揭示了较小片段的核心 RNA 复合物，作为组装由十个 ssRNA 片段组成的完整网络的基础（“锚点”）。在用病毒 RNA 转染的细胞中也鉴定到相同顺序的核心 RNA 复合物形成。这些组装反应不需要病毒蛋白。此外，核心组装体中相互作用碱基的取代突变改变了随后的片段添加，并影响了病毒复制。这些数据确定了一个完全由 RNA 驱动的反应，这可能为设计减毒或抗病毒治疗提供新的机会。
