Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15.
Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15.
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SARS-CoV-2 Nsp15 识别多聚尿苷束的结构基础。
DOI：
10.1101/2023.11.17.567629
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Chen,XiaojiangS
中科院分区：
文献类型：
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作者：
Ito,Fumiaki;Yang,Hanjing;Zhou,ZHong;Chen,XiaojiangS
冠状病毒已经进化出一系列广泛的策略来逃避宿主的抗病毒免疫。当宿主细胞检测到入侵的外源核酸（包括病毒基因组和病毒复制中间体）时，它们会通过细胞质模式识别受体激活干扰素（IFN）信号传导（Kang 等人，2002 年；Kato 等人，2006 年）。冠状病毒表现出的显着隐形活性是由一系列非结构蛋白（Nsps）促进的，例如 Nsp15（Deng 和 Baker，2018；Deng 等人，2019；Hackbart 等人，2020），它是一种尿苷特异性核糖核酸内切酶，可介导逃避宿主对病毒双链 RNA（dsRNA）的检测（Bhardwaj 等人， 2004；Deng 等人，2017；Frazier 等人，2021；Ivano 等人，2004）。 Nsp15 靶向冠状病毒负链 RNA 上的聚尿苷 [poly (U)] 束（Hackbart et al., 2020）。 Nsp15 通过将最初合成的聚 (U) 前导序列修剪至可抑制 dsRNA 形成的最佳长度，限制了负链 RNA 5′ 延伸内聚 (U) 的丰度和长度，但仍可作为冠状病毒正链基因组聚 (A) 尾的模板 (Hofmann 和 Brian，1991)。先前的 Nsp15 结构为其与 ssRNA 和 dsRNA 的结合提供了一些见解（Frazier et al., 2021, 2022; Kim et al., 2020），但 Nsp15 识别聚（U）的精确分子机制仍然不完整。在这里，我们重建了 SARS-CoV-2 Nsp15 与病毒复制 dsRNA 中间体的复合物，该中间体包含病毒基因组的 3ʹ 端，后跟 20 bp 的聚 (A/U) 延伸。低温电子显微镜 (cryoEM) 以 2.3–3.3 Å 分辨率揭示了各种功能状态下的 Nsp15 六聚体结构，包括无 RNA 和两种 dsRNA 结合状态。这些结构的比较表明，Nsp15 六聚体通过与处于两种不同状态的三个亚基直接接触来识别该序列的聚 (U) 段。 Nsp15 的氨基酸残基 W332 和 M330 将靶标尿嘧啶从 dsRNA 的碱基配对中移出，并且移出的碱基被捕获在核酸内切酶活性位点中心。因此，活性位点利用碱基翻转机制将目标尿嘧啶碱基保持在核酸内切酶催化中心以进行切割。 Nsp15 六聚体上锚定有多达 20 个 A/U 碱基对，这解释了与正链中的长聚 (A) 尾相比，负链冠状病毒基因组中聚 (U) 序列大幅缩短的基础。总体而言，我们的结构揭示了 Nsp15 如何与其基因组复制 dsRNA 中间体的聚 (A/U) 序列结合以逃避宿主抗病毒反应。为了了解 SARS-CoV-2 Nsp15 靶向聚（U）的机制，我们重建了 Nsp15 的核糖核蛋白复合物和 35 bp dsRNA 底物，该底物包含 SARS-CoV-2 基因组 3ʹ 端的最后 15 bp 和 20 bp 聚（A/U）延伸，代表冠状病毒基因组复制中间体。
Coronaviruses have evolved a wide array of tactics to evade host antiviral immunity. When host cells detect invading foreign nucleic acids including viral genome and viral replication intermediates, they activate interferon (IFN) signaling via cytoplasmic pattern recognition receptors (Kang et al., 2002; Kato et al., 2006). Remarkable stealth activities exhibited by coronaviruses are facilitated by a series of non-structural proteins (Nsps), such as Nsp15 (Deng and Baker, 2018; Deng et al., 2019; Hackbart et al., 2020), which is a uridine-specific endoribonuclease that mediates evasion of host detection of viral double-stranded RNA (dsRNA)(Bhardwaj et al., 2004; Deng et al., 2017; Frazier et al., 2021; Ivano v et al., 2004). Nsp15 targets a polyuridine [poly (U)] tract on the coronavirus negative strand RNA (Hackbart e t al., 2020). Nsp15 limits the abundance and length of poly (U) within the negative strand RNA 5ʹ-extension by trimming down the initially synthesized poly (U) lead sequence to the optimal length that can suppress dsRNA formation but still serves as a template for poly (A) tail of the positive strand genome of the coronaviruses (Hofmann and Brian, 1991). Prior Nsp15 structures have provided some insights into its binding to both ssRNA and dsRNA (Frazier et al., 2021, 2022; Kim et al., 2020), but the precise molecular mechanisms underlying the recognition of poly (U) by Nsp15 remain incomplete. Here we reconstituted a complex of SARS-CoV-2 Nsp15 with a viral replicative dsRNA intermediate containing 3ʹ-end of the viral genome followed by a 20-bp poly (A/U) extension. Cryogenic electron microscopy (cryoEM) revealed Nsp15 hexamer structures at 2.3–3.3 Å resolution at various functional states, including RNA-free and two dsRNA-bound states. Comparison of these structures shows that the poly (U) tract of the sequence is recognized by an Nsp15 hexamer via direct contact with three subunits in two distinct states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Thus, the active site utilizes a base-flipping mechanism to hold the target uracil base in the endonuclease catalytic center for cleavage. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly (U) sequence in the negative strand coronavirus genome compared to the long poly (A) tail in its positive strand. Overall, our structures reveal how Nsp15 binds to the poly (A/U) sequence of its genomic replicative dsRNA intermediate to evade host antiviral response. To understand the mechanism of poly (U) targeting by SARS-CoV-2 Nsp15, we reconstituted a ribonucleoprotein complex of Nsp15 and a 35-bp dsRNA substrate comprising the final 15-bp of 3ʹ-end of the SARS-CoV-2 genome and 20-bp poly (A/U) extension, which represents a coronavirus genome replication intermediate.