Laboratory of Clinical Research on Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
Center for Vaccine Research and Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA.
Sci Rep. 2024 Aug 23;14(1):19594. doi: 10.1038/s41598-024-70601-5.
Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3' untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.
冠状病毒（CoV）在其正链基因组 RNA 中拥有许多具有功能的顺式作用元件。尽管这些 RNA 结构大多数参与病毒复制，但 CoV 基因组 RNA 中 RNA 结构在病毒复制中的功能仍不清楚。在这项研究中，我们研究了中东呼吸综合征冠状病毒（MERS-CoV）ORF1a 编码区中更高阶 RNA 茎环（SL）结构 SL5B、SL5C 和 SL5D 的功能。我们使用细菌人工染色体系统的反向遗传学方法，揭示了 SL5B 和 SL5C 在 MERS-CoV 的不连续转录中发挥重要作用。计算机分析预测 SL5C 与 3'非翻译区中的一个膨出茎环（BSL）相互作用，表明 SL5C 的 RNA 结构对病毒 RNA 转录很重要。相反，SL5D 不影响转录，但介导正链基因组 RNA 的合成。此外，SL5D 突变体的回复病毒的 SL5 区 RNA 二级结构与野生型相似，表明 SL5D 的 RNA 结构可以精细调节 MERS-CoV 中的 RNA 复制。我们的数据表明，MERS-CoV 基因组 RNA 中的高阶 RNA 结构对病毒 RNA 转录和复制具有新的调控机制。
