The coronavirus replicase complex: structure-function relationship and virus-host interaction

In this project we will provide detailed studies on the structure-function relationship of the coronavirus RTC and link this to virus-host interactions that are decisive for the course of coronavirus infection. We hypothesize that virus-host interactions taking place at, or in close proximity to, the RTC are decisive for host cell innate immune and cytokine responses. In Aim 1 we will therefore assess the cellular microenvironment of the coronavirus RTC by using proximity labelling under various experimental conditions, such as infection with virus mutants that are known to be defective in innate immune evasion, and treatment of cells with interferon (IFN) before infection. We will link those proteome studies with the assessment of the cellular transcriptional response during the virus life cycle to elucidate the kinetics of virus-host interactions at the RTC and the resulting cellular response. In Aim 2 we will perform structural studies to elucidate the overall RTC structure and possible contact sites of (enzymatic) RTC domains and functions with the host cell cytoplasm. We hypothesise that localization of individual viral non-structural proteins may differ and define an RTC component engaged in RNA synthesis and a scaffolding component providing the molecular and membranous environment for RNA synthesis and for virus-host interactions. Studies of aims 1 and 2 will be complemented in Aim 3 by a strong bioinformatics component to identify crucial host cell proteins and pathways that impact virus-host interaction and the cellular response. Finally, in Aim 4 we will assess the consequences of RTC structure and localization on RNA recombination, a process that is driving the evolution and emergence of coronaviruses.