PDF(Pubmed)
Abstract：
There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.
摘要：
关于SARS-CoV-2感染的分子细节仍有许多需要揭示的地方。作为最丰富的蛋白质，冠状病毒核衣壳（N）蛋白衣壳化病毒RNA，作为核糖核蛋白和病毒体的结构成分，并参与转录，复制,和主办条例。病毒与宿主的相互作用可能为更好地了解病毒在感染过程中如何影响或受其宿主的影响以及确定有希望的治疗候选物提供线索。考虑到N的关键作用，我们在这里建立了SARS-CoV-2N的新的细胞相互作用组，通过使用高特异性亲和纯化（S-pulldown）分析与定量质谱和免疫印迹验证，发现许多以前未报道的N相互作用宿主蛋白。生物信息学分析表明,这些宿主因子主要参与翻译调控,病毒转录，RNA过程，应激反应，蛋白质折叠和修饰，和炎症/免疫信号通路，符合N在病毒感染中的假定作用。然后开采现有的药理学细胞靶标和指导药物，产生药物-宿主蛋白网络。因此，我们通过实验确定了几种小分子化合物作为抗SARS-CoV-2复制的新型抑制剂。此外，一个新发现的宿主因素，DDX1被证实主要通过与病毒蛋白的NTD结构域结合而与N相互作用和共定位。重要的是,损失/获得/功能重建实验表明，DDX1是一种有效的抗SARS-CoV-2宿主因子，抑制病毒复制和蛋白质表达。DDX1的N靶向和抗SARS-CoV-2能力始终与其ATPase/解旋酶活性无关。进一步的机制研究表明，DDX1阻碍了N的多种活性，包括N-N相互作用，N低聚,和N-病毒RNA结合，从而可能抑制病毒的繁殖。这些数据为更好地描述N细胞相互作用和SARS-CoV-2感染提供了新的线索，并且可能有助于指导新的治疗候选物的开发。
