Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, Architecture et Réactivité de l'ARN, CNRS UPR9002, 2, allée Konrad Roentgen, F-67084 Strasbourg, France.
Nucleic Acids Res. 2023 Sep 8;51(16):8677-8690. doi: 10.1093/nar/gkad627.
In severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the non-structural protein NSP1 inhibits translation of host mRNAs by binding to the mRNA entry channel of the ribosome and, together with the 5'-untranslated region (UTR) of the viral mRNAs, allows the evasion of that inhibition. Here, we show that NSP1 mediates endonucleolytic cleavages of both host and viral mRNAs in the 5'UTR, but with different cleavage patterns. The first pattern is observed in host mRNAs with cleavages interspersed regularly and close to the 5' cap (6-11 nt downstream of the cap). Those cleavage positions depend more on the position relative to the 5' cap than on the sequence itself. The second cleavage pattern occurs at high NSP1 concentrations and only in SARS-CoV-2 RNAs, with the cleavages clustered at positions 45, 46 and 49. Both patterns of cleavage occur with the mRNA and NSP1 bound to the ribosome, with the SL1 hairpin at the 5' end sufficient to protect from NSP1-mediated degradation at low NSP1 concentrations. We show further that the N-terminal domain of NSP1 is necessary and sufficient for efficient cleavage. We suggest that in the ribosome-bound NSP1 protein the catalytic residues of the N-terminal domain are unmasked by the remodelling of the α1- and α2-helices of the C-terminal domain.
在严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 中，非结构蛋白 NSP1 通过与核糖体的 mRNA 进入通道结合，抑制宿主 mRNAs 的翻译，并与病毒 mRNAs 的 5'-非翻译区 (UTR) 一起，逃避这种抑制。在这里，我们表明 NSP1 介导宿主和病毒 mRNAs 在 5'UTR 中的内切酶切割，但具有不同的切割模式。第一种模式发生在宿主 mRNAs 中，切割位点均匀且靠近 5'帽（帽下游 6-11nt）。这些切割位置更多地取决于相对于 5'帽的位置，而不是序列本身。第二种切割模式发生在高浓度的 NSP1 下，仅在 SARS-CoV-2 RNA 中，切割位点聚集在位置 45、46 和 49。这两种切割模式都发生在与核糖体结合的 mRNA 和 NSP1 上，5'端的 SL1 发夹足以在低浓度的 NSP1 下保护免受 NSP1 介导的降解。我们进一步表明，NSP1 的 N 端结构域对于有效切割是必需和充分的。我们认为，在核糖体结合的 NSP1 蛋白中，N 端结构域的催化残基通过 C 端结构域的α1-和α2-螺旋的重排而暴露。
