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.
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.
Nucleic Acids Res. 2022 Aug 12;50(14):8290-8301. doi: 10.1093/nar/gkac589.
Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.
冠状病毒在病毒复制过程中产生双链 (ds) RNA 中间体，这些中间体可以激活宿主免疫传感器。为了逃避宿主模式识别受体 MDA5 的激活，冠状病毒利用 Nsp15，它是一种尿嘧啶特异性内切核糖核酸酶。据推测，Nsp15 与双层膜囊泡内的冠状病毒复制转录复合物结合，以切割这些 dsRNA 中间体。由于之前对 Nsp15 的结构研究仅限于小的单链 (ss) RNA 底物，因此人们对 Nsp15 如何识别和处理 dsRNA 知之甚少。在这里，我们展示了 SARS-CoV-2 Nsp15 与 52nt dsRNA 结合的冷冻电镜结构。我们观察到 Nsp15 六聚体形成了一个横跨多个原体结合 dsRNA 的平台。这些结构以及定点突变和 RNA 切割实验揭示了对 dsRNA 识别和处理的关键见解。为了处理 dsRNA，Nsp15 利用碱基翻转机制将活性位点内的尿嘧啶正确定向进行切割。我们的研究结果表明，Nsp15 是一种独特的内切核糖核酸酶，能够有效地切割 ssRNA 和 dsRNA。
