Cleavage sequence specificity of Nsp15

Nsp15 is an EndoU nuclease that is partially responsible for SARS-CoV-2’s ability to evade the immune system response. Despite its importance, the sequence specificity of Nsp15 remains difficult to fully determine. In this work, we use a systematic approach to measure Nsp15’s sequence specificity by testing all 16 dinucleotides for cleavage activity. The results show a preference for uridine in the first dinucleotide position, but with varying specificity in the second position. Using Alphafold3 predictions to examine the structural basis of this specificity suggests important contacts 3’ of the dinucleotide sequence as well as contacts to the dinucleotides that agree with the cleavage specificity.

非结构蛋白15（Nsp15）是一种内切U核酸酶（EndoU nuclease），在严重急性呼吸综合征冠状病毒2型（SARS-CoV-2，即新冠病毒）逃避免疫应答的过程中发挥了部分作用。尽管该蛋白功能至关重要，但目前仍难以完全阐明Nsp15的序列特异性。本研究采用系统性研究策略，通过检测全部16种二核苷酸（dinucleotide）的切割活性，对Nsp15的序列特异性进行定量表征。实验结果显示，Nsp15对二核苷酸第一位的尿苷（uridine）具有显著偏好性，但在第二位核苷酸位点上则表现出差异化的序列特异性。本研究借助AlphaFold3（阿尔法折叠3）的预测结果解析该特异性的结构基础，结果表明该蛋白不仅与二核苷酸序列的3'端存在关键相互作用，还可与符合其切割特异性的二核苷酸形成特异性结合。