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

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 its 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.This proposal assembles an interdisciplinary team by joining the forces of five labs that combine expertise in diverse areas including molecular virology of coronaviruses, translation mechanisms (including that of viral RNAs), RNA modifications, codon pair deoptimization strategies, high-throughput sequencing, and different animal models for pathogenicity, transmission and immunogenicity. Importantly, our multi-faceted approach will allow us to go beyond the current state of the art, in particular since this team has direct access to live virus samples, various animal models, BSL3 laboratories and animal facilities, and the ability to create recombinant SARS-CoV-2 for experimentation. We are confident that the combined knowledge generated on this new virus can rapidly facilitate vaccine development.Importantly, we will make our initial ribosome profiling and RNA methylation data available immediately after acquisition to speed up research during this ongoing crisis.