A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.
A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.
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DOI：
10.1128/jvi.01678-21
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发表时间：
2022-01-26
影响因子：
5.4
通讯作者：
Mazumder B
中科院分区：
文献类型：
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作者：
Basu A;Penumutchu S;Nguyen K;Mbonye U;Tolbert BS;Karn J;Komar AA;Mazumder B
SARS-CoV-2正向单链RNA基因组含有重要的顺式作用元件，控制病毒基因表达的关键方面。然而，关于这些元件如何感知宿主细胞的各种信号并调节病毒蛋白质合成的见解尚不清楚。在这里，我们在SARS-CoV-2ORF1a和S RNA中发现了两个新的顺式调控元件，并描述了它们在SARS-CoV-2翻译控制中的作用。这些元件是序列无关的，但形成保守的发夹结构(经核磁共振验证)，类似于伽马激活的翻译抑制(GATT)元件，在一组人mRNAs中发现，这些元件指导对干扰素-γ反应的髓系细胞的翻译抑制。我们的研究表明，用受体结合的S1亚基、S蛋白假型慢病毒和含S蛋白的病毒样颗粒处理人肺细胞，可以触发一条涉及DAP-kinase1的信号通路，导致核糖体蛋白L13a的磷酸化和从大的核糖体亚基释放。释放的L13a形成病毒激活的翻译抑制因子(VAIT)复合体，与ORF1a和S VIFT元件结合，导致翻译沉默。翻译沉默需要细胞外的S蛋白(及其与宿主ACE2受体的相互作用)，而不是细胞内的合成。RNA-蛋白质相互作用分析和体外翻译实验表明，步态和VAIT元件不相互竞争，突显了两条途径之间的差异。SARS-CoV-2基因组的序列比对显示了Vait元件的高度保守性，表明它们具有重要的功能。这种Vait介导的SARS-CoV-2翻译调控机制可能为小分子干预提供新的靶点和/或促进更有效的mRNA疫苗的开发。重要的是，RNA病毒基因组中特定的RNA元件在宿主与病毒的相互作用中发挥着重要作用。对于SARS-CoV-2，缺乏关于这些RNA元件如何感知宿主细胞信号的机械性见解。在这里，我们报告了步态样SARS-CoV-2RNA元件(称为VAITs)与宿主细胞产生的信号之间的一种新的关系。我们发现，对于SARS-CoV-2，Spike蛋白与ACE2的相互作用不仅用于病毒进入宿主细胞的目的，而且还传递最终导致L13a从大的核糖体亚基磷酸化和释放的信号。我们还表明，这一事件导致ORF1a和S mRNAs的翻译停滞，这种方式取决于RNA元件的结构。由病毒蛋白触发的宿主细胞产生的信号对病毒mRNA的翻译调控是宿主与病毒关系中的一种新范式。
The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.