Shuvalov Alexey, Shuvalova Ekaterina, Biziaev Nikita, Sokolova Elizaveta, Evmenov Konstantin, Pustogarov Nikolay, Arnautova Aleksandra, Matrosova Vera, Egorova Tatiana, Alkalaeva Elena
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
RNA Biol. 2021 Nov 12;18(sup2):804-817. doi: 10.1080/15476286.2021.1999103. Epub 2021 Nov 18.
Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.
新型冠状病毒（SARS-CoV-2）的Nsp1可调节细胞中宿主和病毒mRNA的翻译。Nsp1通过封闭40S核糖体亚基的进入通道来抑制宿主翻译起始。已报道的Nsp1-核糖体复合物的结构研究显示，终止后80S复合物包含Nsp1、eRF1和ABCE1。鉴于Nsp1存在于终止后80S核糖体复合物中，我们推测Nsp1可能参与翻译终止过程。通过使用无细胞翻译系统和重组翻译系统，我们发现Nsp1可刺激肽段释放并促进终止复合物的形成。对翻译终止阶段Nsp1活性的详细分析表明，Nsp1有助于终止密码子的识别。我们证明Nsp1的刺激作用靶向eRF1，而不影响eRF3。此外，Nsp1可增加所有三个终止密码子处终止复合物的数量。Nsp1在翻译终止中的活性由其N端结构域提供，而eRF1的最小必需部分是NM结构域。我们推测Nsp1在翻译终止中发挥作用的生物学意义在于与正在翻译宿主mRNA的80S核糖体结合，并将它们从活跃核糖体池中清除。
