V. A. GREEN¹, L. PELKMANS<sup>1

1</sup>Institute of Molecular Life Sciences, University of Zurich, Winterhurerstrasse 190, Zurich 8057, Switzerland

Pathogen invasion is often accompanied by widespread alterations in cellular physiology, which reflects the hijacking of host factors and processes for entry and replication. Although genetic perturbation screens have revealed the complexity of host factors involved for numerous pathogens, it has remained challenging to disentangle this complexity along the progression of host cell reorganisation during the infection process. We here address this by combining the first high confidence, image-based, genome-scale RNAi screening of rotavirus infection in human intestinal cells with an innovative approach to infer a continuous trajectory of virus infection progression from fixed cell populations. We uncover a complex, yet ordered host cell reorganisation program during rotavirus infection that provides a replication-permissive cellular environment for the virus. This includes an alternative mechanism for host protein synthesis shut-off during early stages of infection, consumption of lipid stores, rearrangement of mitochondria, and ER shape remodelling throughout infection, modulated by the ER shaping protein REEP2. Finally, by integrating the large-scale gene perturbation dataset with single-cell trajectories, we propose a model for host factor-mediated cellular reorganisation during rotavirus infection, many elements of which can be linked to calcium-activated AMPK signalling. Our work provides a powerful approach to order the complexity of host cellular requirements along a trajectory of cellular reorganisation during pathogen invasion, an approach that would enhance the analysis of perturbation screens from any field.