Translational Regulation of SARS-CoV-2 in response to viral S protein-induced signaling

RNA病毒基因组内RNA元件的采用折叠允许宿主和病毒的结合。 蛋白质，并在宿主-病毒相互作用中发挥重要作用。然而，关于这些机制如何 SARS冠状病毒2缺乏能感受宿主细胞信号转导的RNA元件。这个项目是 基于我们最近在ORF1a RNA中发现了一种新的结构保守的RNA元件， SARS-CoV-2的S mRNA。我们的研究表明，这种元件调节病毒蛋白的翻译， 对病毒S蛋白诱导的人肺细胞信号的反应。在先前的研究中，我们发现了一个翻译的 沉默依赖性机制控制骨髓细胞炎症。这些研究表明， L13 a依赖性多蛋白质RNA结合复合物（IFN-γ激活的抑制剂）的组装 翻译的3 '非翻译区（UTR）中发现的GAIT元件上的GAIT复合物 引起翻译抑制的mRNA。我们的研究还表明， 作为对炎症的内源性防御机制。GAIT样因子在人体内的重要性 对冠状病毒和其他呼吸道病原体的反应是从我们发表的研究中出现的， 他人我们在该提案中提出的初步工作表明，用SARS治疗人肺细胞- CoV-2 S蛋白激活DAP激酶依赖性信号传导机制，导致L13 a从 “GAIT样”病毒上核糖体大亚基随后形成L13 a依赖性RNA结合复合物 SARS-CoV-2的RNA元件，从而抑制翻译。这让我们将这些命名为 病毒激活的翻译抑制因子（VAIT）。总之，这些研究使我们 假设SARS-CoV-2感染后核糖体释放形式L13 a促进RNA- ORF1a RNA和S mRNA的VAIT元件上的结合复合物，其在生物化学和功能上不同 来自IFN-g诱导的GAIT复合物。由VAIT复合物引起的翻译抑制作用是可测试的。 我们将通过以下两个目标来检验这一假设。目标1。为了阐明 翻译抑制性VAIT RNP复合物和信号传导中上游激酶的鉴定。为此，我们 将使用凝胶过滤、RNA亲和色谱和质谱的组合方法， 鉴定RNA结合复合物的亚基。潜在激酶的小分子抑制剂和siRNA 将用于剖析信号方面。目标二。验证VAIT元件的病毒学意义 介导的翻译沉默。为此，我们将在S基因的VAIT元件中使用广泛的突变， 在反式互补系统的病毒基因组RNA内，以测试VAIT元件介导的 在病毒蛋白和核酸的细胞内表达中的翻译沉默。机械的见解， VAIT元件介导的SARS-CoV-2 RNA的翻译沉默可能提供新的靶点， 分子干预和促进下一代mRNA疫苗的开发。

Adopted folding of the RNA elements within the genomes of RNA viruses allows binding of host and viral proteins and plays an essential role in host-virus interactions. However, the mechanistic insights on how these RNA elements could sense the transduced signals from host cells are lacking for SARS-CoV-2. This project is based on our recent discovery of a novel and structurally conserved RNA element within the ORF1a RNA and S mRNA of SARS-CoV-2. Our studies showed that this element regulates the translation of viral proteins in response to viral S protein-induced signaling in human lung cells. In prior studies, we identified a translational silencing-dependent mechanism of controlling inflammation in myeloid cells. These studies showed that the assembly of an L13a-dependent multi-protein RNA-binding complex (IFN-gamma-activated-inhibitor of translation) or GAIT complex on the GAIT elements found in the 3' untranslated region (UTR) of target mRNAs causing translational inhibition. Our studies also showed the physiological importance of this mechanism as an endogenous defense against inflammation. Importance of the GAIT-like elements in human responses to coronavirus and other respiratory pathogens is emerging from the published studies by us and others. Our preliminary work presented in this proposal shows that treatment of human lung cells with SARS- CoV-2 S protein activates a DAP kinase-dependent signaling mechanism causing the release of L13a from large ribosomal subunit followed by the formation of L13a-dependent RNA-binding complex on “GAIT-like” viral RNA elements of SARS-CoV-2, thus inhibiting translation. This led us to name these elements virus activated inhibitor of translation (VAIT) elements. Together, these studies lead us to hypothesize that ribosome-released form of L13a upon SARS-CoV-2 infection promotes the assembly of RNA- binding complex on VAIT element of ORF1a RNA and S mRNA, which is biochemically and functionally distinct from IFN-g-induced GAIT complex. The role of translational inhibition caused by the VAIT complex is testable. We will test this hypothesis by pursuing the following two aims. Aim 1. To elucidate the composition of the translation inhibitory VAIT RNP complex and identification of the upstream kinase in signaling. In this aim we will use the combined approach of Gel filtration, RNA-affinity chromatography and mass spectrometry to identify the subunits of the RNA-binding complex. Small molecule inhibitors and siRNA of the potential kinases will be used to dissect the signaling aspect. Aim 2. To test the virological significance of VAIT element mediated translational silencing. In this aim we will use extensive mutation in the VAIT element of the S gene within the viral genomic RNA of the trans-complementation system to test the role of VAIT element-mediated translational silencing in the intracellular expression of viral proteins and nucleic acid. Mechanistic insights on VAIT element mediated translational silencing of SARS-CoV-2 RNAs may provide novel targets of small molecule intervention and facilitate the development of next-generation mRNA vaccines.