Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA.
Nucleic Acids Res. 2024 Nov 27;52(21):13419-13433. doi: 10.1093/nar/gkae939.
Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology.
冠状病毒内切核糖核酸酶 Nsp15 通过抑制病毒 dsRNA 水平来逃避宿主先天免疫。Nsp15 在体外对 ssRNA 和 dsRNA 均具有强烈的偏好，优先切割未配对或凸起的 U 残基，其活性受到二价离子的刺激。在这里，我们系统地量化了定义其特异性的 RNA 序列和结构环境的影响。结果表明，之前观察到的对 U↓A/G 的序列偏好仅对 kcat/Km 贡献约 2 倍。相比之下，dsRNA 结构中凸起 U 残基侧翼的序列与 ssRNA 相比将 kcat/Km 提高了一个数量级，而 dsRNA 中的碱基配对则基本阻断了切割。尽管多次转化动力学存在巨大差异，但 RNA 结构对切割步骤的影响很小。令人惊讶的是，尽管二价离子对 Nsp15 的激活被广泛认为对其生物学功能很重要，但对 kcat/Km 的影响仅约为 2 倍，且与 RNA 结构无关。这些结果揭示了由 RNA 结构主导的特异性景观，并为识别特异性的基础相互作用、确定抑制和抗性机制以及定义对冠状病毒生物学重要的靶标提供了定量框架。
