Life Sciences Institute, Zhejiang University , Hangzhou, Zhejiang, China.
Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University , Hangzhou, Zhejiang, China.
mSystems. 2023 Aug 31;8(4):e0013523. doi: 10.1128/msystems.00135-23. Epub 2023 Jun 14.
A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions is crucial to developing effective therapeutics and addressing the threat of emerging coronaviruses. The role of noncoding regions of viral RNA (ncrRNAs) has yet to be systematically scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry and designed a diverse set of bait ncrRNAs to systematically map the interactome of SARS-CoV-2 ncrRNA in Calu-3, Huh7, and HEK293T cells. Integration of the results defined the core ncrRNA-host protein interactomes among cell lines. The 5' UTR interactome is enriched with proteins in the small nuclear ribonucleoproteins family and is a target for the regulation of viral replication and transcription. The 3' UTR interactome is enriched with proteins involved in the stress granules and heterogeneous nuclear ribonucleoproteins family. Intriguingly, compared with the positive-sense ncrRNAs, the negative-sense ncrRNAs, especially the negative-sense of 3' UTR, interacted with a large array of host proteins across all cell lines. These proteins are involved in the regulation of the viral production process, host cell apoptosis, and immune response. Taken together, our study depicts the comprehensive landscape of the SARS-CoV-2 ncrRNA-host protein interactome and unveils the potential regulatory role of the negative-sense ncrRNAs, providing a new perspective on virus-host interactions and the design of future therapeutics. Given the highly conserved nature of UTRs in positive-strand viruses, the regulatory role of negative-sense ncrRNAs should not be exclusive to SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic affecting millions of lives. During replication and transcription, noncoding regions of the viral RNA (ncrRNAs) may play an important role in the virus-host interactions. Understanding which and how these ncrRNAs interact with host proteins is crucial for understanding the mechanism of SARS-CoV-2 pathogenesis. We developed the MS2 affinity purification coupled with liquid chromatography-mass spectrometry method and designed a diverse set of ncrRNAs to identify the SARS-CoV-2 ncrRNA interactome comprehensively in different cell lines and found that the 5' UTR binds to proteins involved in U1 small nuclear ribonucleoprotein, while the 3' UTR interacts with proteins involved in stress granules and the heterogeneous nuclear ribonucleoprotein family. Interestingly, negative-sense ncrRNAs showed interactions with a large number of diverse host proteins, indicating a crucial role in infection. The results demonstrate that ncrRNAs could serve diverse regulatory functions.
深入了解严重急性呼吸系统综合症冠状病毒 2(SARS-CoV-2)与宿主的相互作用对于开发有效的治疗方法和应对新兴冠状病毒的威胁至关重要。病毒 RNA 的非编码区域(ncrRNAs)的作用尚未得到系统研究。我们开发了一种使用 MS2 亲和纯化结合液相色谱-质谱联用的方法,并设计了一系列多样化的诱饵 ncrRNAs,以系统地绘制 SARS-CoV-2 ncrRNA 在 Calu-3、Huh7 和 HEK293T 细胞中的互作组图谱。整合结果定义了细胞系之间的核心 ncrRNA-宿主蛋白互作组。5'UTR 互作组富含小核核糖核蛋白家族的蛋白质,是调节病毒复制和转录的靶点。3'UTR 互作组富含参与应激颗粒和异质核核糖核蛋白家族的蛋白质。有趣的是,与正链 ncrRNAs 相比,负链 ncrRNAs,特别是 3'UTR 的负链,与所有细胞系中的大量宿主蛋白相互作用。这些蛋白质参与病毒产生过程、宿主细胞凋亡和免疫反应的调节。总的来说,我们的研究描绘了 SARS-CoV-2 ncrRNA-宿主蛋白互作组的全面图谱,并揭示了负链 ncrRNAs 的潜在调节作用,为病毒-宿主相互作用和未来治疗方法的设计提供了新的视角。鉴于正链病毒 UTR 的高度保守性,负链 ncrRNAs 的调节作用不应仅限于 SARS-CoV-2。
严重急性呼吸系统综合症冠状病毒 2(SARS-CoV-2)导致 COVID-19,这是一种影响数百万人生命的大流行。在复制和转录过程中,病毒 RNA 的非编码区域(ncrRNAs)可能在病毒-宿主相互作用中发挥重要作用。了解这些 ncrRNAs 与宿主蛋白相互作用的方式和方式对于理解 SARS-CoV-2 发病机制至关重要。我们开发了 MS2 亲和纯化结合液相色谱-质谱联用的方法,并设计了一系列多样化的 ncrRNAs,以全面鉴定不同细胞系中 SARS-CoV-2 ncrRNA 的互作组,发现 5'UTR 与 U1 小核核糖核蛋白相关蛋白结合,而 3'UTR 与应激颗粒和异质核核糖核蛋白家族相关蛋白相互作用。有趣的是,负链 ncrRNAs 与大量不同的宿主蛋白相互作用,表明其在感染过程中起着关键作用。结果表明,ncrRNAs 可能具有多种调节功能。