Recoding the SARS-CoV-2 genome - A multidisciplinary approach to generate live-attenuated coronavirus vaccines

新发现的冠状病毒（CoV）SARS-CoV-2是最近威胁地球仪无数生命的上呼吸道疾病和肺炎大流行的罪魁祸首。像所有病毒一样，它严重依赖于细胞代谢的重新编程，特别是劫持宿主的翻译机制。该提案的目标是确定病毒在侵占宿主细胞期间的脆弱性。具体来说，我们将全面测试SARS-CoV-2可能用来劫持宿主翻译的多个方面。我们将利用这些知识来合理设计重新编码的冠状病毒基因组，以开发新型冠状病毒减毒活疫苗。这一策略不仅适用于该病毒，也适用于未来新出现的人畜共患病病毒。因此，我们首先要问的是，该病毒是否劫持了宿主的RNA修饰机制来修饰自己的RNA基因组，以避免被宿主细胞的先天免疫防御系统检测到。其次，我们将确定介导病毒基因组修饰的宿主RNA修饰机制。第三，我们将研究病毒RNA修饰是否促进宿主翻译机制的募集。为此，我们将在高分辨率感染时程中使用核糖体分析和RNAseq来定量确定宿主细胞的翻译反应。这将揭示SARS-CoV-2在其生命周期中如何利用宿主的mRNA翻译机制。第四，我们将测试病毒是否调节tRNA和tRNA修饰的水平，以实现有效的翻译，尽管其基因组和宿主基因组之间的密码子使用不同。第五，我们将应用所获得的知识开发一系列合成减毒病毒，例如缺乏RNA修饰或含有在感染期间难以翻译的序列元件。我们将在体外和体内分析产生的病毒构建体，并通过核糖体分析和动物模型测试选定的病毒。通过结合这些方法，我们将确定SARS-CoV-2如何与其宿主相互作用，特别是其翻译和RNA修饰机制。这将鉴定减毒病毒变体以及药物靶点和策略，以合理设计可用于疫苗开发的减毒病毒，也可用于其他病毒。

The newly discovered coronavirus (CoV) SARS-CoV-2 is responsible for the recent pandemic of upper respiratory disease and pneumonia that threatens countless lives across the globe. Like all viruses it critically relies on reprogramming of the cellular metabolism, in particular on hijacking the translation machinery of its host. The goal of this proposal is to identify vulnerabilities of the virus during its usurpation of the host cell. Specifically, we will comprehensively test multiple aspects that SARS-CoV-2 may use to hijack host translation. We will use this knowledge to rationally design re-coded coronavirus genomes in order to develop novel live-attenuated coronavirus vaccines. This strategy will not only be applicable for this virus but also for newly emerging zoonotic viruses in the future.Therefore, we will first ask whether the virus hijacks the host RNA modifications machinery to modify its own RNA genome to avoid detection by the host cell’s innate immune defense systems. Second, we will identify the host RNA modification machinery that mediates the modification of the viral genome. Third, we will examine whether viral RNA modifications facilitate the recruitment of the host translation machinery. To this end, we will use ribosome profiling and RNAseq in a high-resolution infection time course to quantitatively determine the translational response of the host cell. This will reveal how SARS-CoV-2 exploits the mRNA translation machinery of the host during its life cycle. Fourth, we will test whether the virus modulates the levels of tRNA and tRNA modifications to achieve efficient translation despite the diverging codon usage between its genome and the one of its host. Fifth, we will apply the knowledge gained to develop a series of synthetic attenuated viruses lacking, for example, RNA modifications or containing sequence elements that are difficult to translate during an infection. We will analyze the generated virus constructs both in vitro and in vivo and test selected viruses by ribosome profiling and in animal models. By combining these approaches, we will identify how SARS-CoV-2 interacts with ist host and in particular its translation and RNA modification machineries. This will identify attenuated virus variants as well as drug targets and strategies to rationally design attenuated viruses that can be used for vaccine development also for other viruses.