Department of Microbiology and Immunology, UNC-Chapel Hill, Chapel Hill, North Carolina, USA.
Department of Genetics, UNC-Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2020 Nov 23;94(24). doi: 10.1128/JVI.00701-20.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis. Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.
基孔肯雅病毒（CHIKV）是一种虫媒传播的甲病毒，与人类的关节疼痛有关。病毒基因组中的 RNA 二级结构在甲病毒的生命周期中起着重要作用；然而，RNA 结构在调节 CHIKV 复制中的具体作用还知之甚少。我们之前的研究发现，甲病毒之间的 RNA 二级结构几乎没有保守性，这种结构的差异在特定甲病毒基因组中创造了独特的功能结构。因此，为了了解 RNA 结构对 CHIKV 生物学的影响，我们使用 SHAPE-MaP 来推断 2013 年加勒比地区爆发的 CHIKV 分离株基因组中 RNA 二级结构的建模。然后，我们分析了基因组中具有高水平结构特异性的区域，以确定潜在的功能 RNA 二级结构，并在 CHIKV 基因组中鉴定了 23 个具有高于平均结构稳定性的区域，包括之前鉴定的四个具有重要功能的 CHIKV RNA 结构。我们还分析了已知影响蚊子细胞中病毒复制的多个 CHIKV 3'UTR 变体的 RNA 灵活性和二级结构。这项分析在这些 3'UTR 变体中发现了一些新的 RNA 结构。在 3'UTR 中发生的增强蚊子细胞中病毒复制的重复导致 3'UTR 中未折叠 RNA 的总量增加。这项分析表明，CHIKV 基因组包含许多独特的、特定的 RNA 二级结构，并为测试这些二级结构在 CHIKV 复制和发病机制中的功能重要性提供了一种策略。基孔肯雅病毒（CHIKV）是一种虫媒传播的 RNA 病毒，可引起人类发热和关节疼痛。CHIKV 会引起暴发性爆发，但目前尚无批准的疗法来治疗或预防 CHIKV 感染。CHIKV 基因组包含对病毒复制至关重要的功能性 RNA 二级结构。由于仅定义了 CHIKV 基因组的一小部分 RNA 二级结构，我们使用化学探测方法来定义 CHIKV 基因组 RNA 的 RNA 二级结构。我们鉴定了基因组中 23 个高度特异的结构区域，并通过突变来确认一个结构的功能重要性。此外，我们还定义了三种在蚊子细胞中复制能力不同的 CHIKV 3'UTR 变体的 RNA 二级结构。我们的研究强调了 CHIKV 基因组的复杂性，并描述了新的系统来设计补偿突变，以测试病毒 RNA 二级结构的功能相关性。
