Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA. Electronic address: https://twitter.com/@ishamyana.
Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, USA; Department of Chemistry and Biochemistry, College of Charleston, Charleston, SC, 29424, USA(†). Electronic address: https://twitter.com/@MNFrazier5.
J Mol Biol. 2022 Oct 30;434(20):167796. doi: 10.1016/j.jmb.2022.167796. Epub 2022 Aug 19.
Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.
正在进行的 COVID-19 大流行（由新型冠状病毒 SARS-CoV-2 引起）的全球测序工作，继续深入了解病毒基因组的进化。冠状病毒在其基因组的前三分之二编码 16 种非结构蛋白，这些蛋白有助于病毒复制和转录，以及逃避宿主免疫反应。然而，许多这些病毒蛋白仍未得到充分研究。Nsp15 是一种在所有冠状病毒中保守的尿嘧啶特异性内切核糖核酸酶。Nsp15 的核酸酶活性有助于病毒逃避触发先天免疫反应。了解 Nsp15 在大流行过程中的变化方式，以及突变如何影响其 RNA 加工功能，将深入了解依赖寡聚化的内切核糖核酸酶的进化，并为药物设计提供信息。结合先前的结构数据，对 190 多万个 SARS-CoV-2 序列进行的生物信息学分析揭示了 Nsp15 的三个结构域（N 端、中间、内切酶 U）的突变。对选定的 Nsp15 变体进行了生化表征，并与野生型 Nsp15 进行了比较。我们发现，对重要催化残基的突变降低了切割活性，但增加了重组蛋白的六聚体/单体比。我们分析的许多高度流行的变体导致核酸酶活性降低，同时增加了无活性的单体形式。总的来说，我们的工作确定了在患者样本中观察到的 Nsp15 变体如何影响核酸酶活性和寡聚化，深入了解了这些变体在体内的影响。
