Wright Zoe M, Butay Kevin John, Krahn Juno M, Wilson Isha M, Gabel Scott A, DeRose Eugene F, Hissein Israa S, Williams Jason G, Borgnia Mario J, Frazier Meredith N, Mueller Geoffrey A, Stanley Robin E
Molecular and Cellular Biology 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.
Genome Integrity and Structural Biology 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.
Nat Commun. 2025 Jan 4;16(1):391. doi: 10.1038/s41467-024-55682-0.
Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3' of uridine (U). While prior structural data shows that to cleave double stranded RNA, Nsp15's target U must be flipped out of the helix, it is not yet understood whether Nsp15 initiates flipping or captures spontaneously flipped bases. We address this gap by designing fluorinated double stranded RNA substrates that allow us to directly relate a U's sequence context to both its tendency to spontaneously flip and its susceptibility to cleavage by Nsp15. Through a combination of nuclease assays, F NMR spectroscopy, mass spectrometry, and single particle cryo-EM, we determine that Nsp15 acts most efficiently on unpaired Us, particularly those that are already flipped. Across sequence contexts, we find Nsp15's cleavage efficiency to be directly related to that U's tendency to spontaneously flip. Overall, our findings unify previous characterizations of Nsp15's cleavage preferences, and suggest that activity of Nsp15 during infection is partially driven by bulged or otherwise relatively accessible Us that appear at strategic positions in the viral RNA.
冠状病毒借助核糖核酸内切酶Nsp15逃避免疫系统的检测，该酶通过切割尿苷（U）的3'端来调节病毒双链RNA的水平。虽然先前的结构数据表明，为了切割双链RNA，Nsp15的靶标U必须从螺旋中翻转出来，但目前尚不清楚Nsp15是启动翻转还是捕获自发翻转的碱基。我们通过设计氟化双链RNA底物来填补这一空白，这些底物使我们能够直接将U的序列背景与其自发翻转的倾向以及被Nsp15切割的敏感性联系起来。通过核酸酶测定、F NMR光谱、质谱和单颗粒冷冻电镜的组合，我们确定Nsp15对未配对的U作用最有效，特别是那些已经翻转的U。在不同的序列背景下，我们发现Nsp15的切割效率与该U自发翻转的倾向直接相关。总体而言，我们的发现统一了先前对Nsp15切割偏好的表征，并表明Nsp15在感染期间的活性部分由出现在病毒RNA战略位置的凸起或其他相对易接近的U驱动。
