The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.
The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.
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HCoV-OC43的5‘非编码区采用了一种拓扑约束结构来本质上抑制翻译。
DOI：
10.1016/j.jbc.2023.103028
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发表时间：
2023-04
影响因子：
4.8
通讯作者：
Tolbert, Blanton S.
中科院分区：
文献类型：
--
作者：
Mackeown, Matthew;Kung, Yu-An;Davila-Calderon, Jesse;Ford, William P.;Luo, Le;Henry, Barrington;Li, Mei-Ling;Brewer, Gary;Shih, Shin-Ru;Tolbert, Blanton S.
导致COVID-19大流行的SARS-CoV-2的出现突出了快速表征与细胞发病机制相关的病毒机制的必要性。病毒UTR代表有助于这种机制的保守基因组元件。然而，大多数CoV UTR的结构细节不可用。需要实验方法来允许容易地生成高质量的病毒RNA三级结构模型，这可以促进比较机制的努力。通过整合实验和计算技术，我们在此报告了HCoV-OC 43基因组（一种实验室易处理的模型冠状病毒）5′UTR内保守RNA结构的有效表征。我们提供的证据表明，5′非翻译区折叠成一个结构，具有明确的茎环（SL）确定的化学探测和直接检测的氢键核磁共振。我们结合联合收割机实验碱基对的限制与全球结构信息SAXS生成一个三维模型，揭示了SL 1 -4采用拓扑约束的结构，其中SL 3和4同轴堆叠。同轴堆叠由短接头核苷酸介导，并允许SL 1至2通过围绕SL 3，4螺旋轴枢转来采样不同的共接合方向。为了评估SL 3，4同轴螺旋的功能相关性，我们设计了携带HCoV-OC 43 5′UTR的荧光素酶报告基因构建体，其具有设计用于消除同轴堆叠的突变。我们的研究结果表明，SL 3，4螺旋内在地抑制翻译效率，因为不稳定的突变与相对于野生型增加的荧光素酶表达相关，而不影响报告基因mRNA水平，从而突出了5′UTR结构如何有助于病毒机制。
The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5′UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5′UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5′UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5′UTR structure contributes to the viral mechanism.
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病毒粒子内 SARS-CoV-2 RNA 基因组的结构
影响因子：
16.6
作者：
Cao C;Cai Z;Xiao X;Rao J;Chen J;Hu N;Yang M;Xing X;Wang Y;Li M;Zhou B;Wang X;Wang J;Xue Y
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DOI：
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发表时间：
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期刊：
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